Structure-Based and Knowledge-Informed Design of B-Raf Inhibitors Devoid of Deleterious PXR Binding.

Dabrafenib is an anticancer drug currently used in the clinics, alone or in combination. However, dabrafenib was recently shown to potently activate the human nuclear receptor pregnane X receptor (PXR). PXR activation increases the clearance of various chemicals and drugs, including dabrafenib itself. It may also enhance cell proliferation and tumor aggressiveness. Therefore, there is a need for rational design of a potent protein kinase B-Raf inhibitor devoid of binding to the secondary target PXR and resisting rapid metabolism. By determining the crystal structure of dabrafenib bound to PXR and analyzing its mode of binding to both PXR and its primary target, B-Raf-V600E, we were able to derive new compounds with nanomolar activity against B-Raf and no detectable affinity for PXR. The crystal structure of B-Raf in complex with our lead compound revealed a subdomain swapping of the activation loop with potentially important functional implications for a prolonged inhibition of B-Raf-V600E.

Mechanistic insights into the synergistic activation of the RXR-PXR heterodimer by endocrine disruptor mixtures.

Humans are chronically exposed to mixtures of xenobiotics referred to as endocrine-disrupting chemicals (EDCs). A vast body of literature links exposure to these chemicals with increased incidences of reproductive, metabolic, or neurological disorders. Moreover, recent data demonstrate that, when used in combination, chemicals have outcomes that cannot be predicted from their individual behavior. In its heterodimeric form with the retinoid X receptor (RXR), the pregnane X receptor (PXR) plays an essential role in controlling the mammalian xenobiotic response and mediates both beneficial and detrimental effects. Our previous work shed light on a mechanism by which a binary mixture of xenobiotics activates PXR in a synergistic fashion. Structural analysis revealed that mutual stabilization of the compounds within the ligand-binding pocket of PXR accounts for the enhancement of their binding affinity. In order to identify and characterize additional active mixtures, we combined a set of cell-based, biophysical, structural, and in vivo approaches. Our study reveals features that confirm the binding promiscuity of this receptor and its ability to accommodate bipartite ligands. We reveal previously unidentified binding mechanisms involving dynamic structural transitions and covalent coupling and report four binary mixtures eliciting graded synergistic activities. Last, we demonstrate that the robust activity obtained with two synergizing PXR ligands can be enhanced further in the presence of RXR environmental ligands. Our study reveals insights as to how low-dose EDC mixtures may alter physiology through interaction with RXR-PXR and potentially several other nuclear receptor heterodimers.

Crystal Structure of the Carboxy-Terminal Region of the Bacteriophage T4 Proximal Long Tail Fiber Protein Gp34.

Long tail fibers of bacteriophage T4 are formed by proteins gp34, gp35, gp36, and gp37, with gp34 located at the phage-proximal end and gp37 at the phage-distal, receptor-binding end. We have solved the structure of the carboxy-terminal region of gp34, consisting of amino acids 894-1289, by single-wavelength anomalous diffraction and extended the structure to amino acids 744-1289 using data collected from crystals containing longer gp34-fragments. The structure reveals three repeats of a mixed α-ß fibrous domain in residues 744 to 877. A triple-helical neck connects to an extended triple ß-helix domain (amino acids 900-1127) punctuated by two ß-prism domains. Next, a ß-prism domain decorated with short helices and extended ß-helices is present (residues 1146-1238), while the C-terminal end is capped with another short ß-helical region and three ß-hairpins. The structure provides insight into the stability of the fibrous gp34 protein.

Structure of the Receptor-Binding Carboxy-Terminal Domain of the Bacteriophage T5 L-Shaped Tail Fibre with and without Its Intra-Molecular Chaperone.

Bacteriophage T5, a Siphovirus belonging to the order Caudovirales, has a flexible, three-fold symmetric tail, to which three L-shaped fibres are attached. These fibres recognize oligo-mannose units on the bacterial cell surface prior to infection and are composed of homotrimers of the pb1 protein. Pb1 has 1396 amino acids, of which the carboxy-terminal 133 residues form a trimeric intra-molecular chaperone that is auto-proteolyzed after correct folding. The structure of a trimer of residues 970-1263 was determined by single anomalous dispersion phasing using incorporated selenomethionine residues and refined at 2.3 Å resolution using crystals grown from native, methionine-containing, protein. The protein inhibits phage infection by competition. The phage-distal receptor-binding domain resembles a bullet, with the walls formed by partially intertwined beta-sheets, conferring stability to the structure. The fold of the domain is novel and the topology unique to the pb1 structure. A site-directed mutant (Ser1264 to Ala), in which auto-proteolysis is impeded, was also produced, crystallized and its 2.5 Å structure solved by molecular replacement. The additional chaperone domain (residues 1263-1396) consists of a central trimeric alpha-helical coiled-coil flanked by a mixed alpha-beta domain. Three long beta-hairpin tentacles, one from each chaperone monomer, extend into long curved grooves of the bullet-shaped domain. The chaperone-containing mutant did not inhibit infection by competition.

The Melibiose Transporter of Escherichia coli: CRITICAL CONTRIBUTION OF LYS-377 TO THE STRUCTURAL ORGANIZATION OF THE INTERACTING SUBSTRATE BINDING SITES.

We examine the role of Lys-377, the only charged residue in helix XI, on the functional mechanism of the Na(+)-sugar melibiose symporter from Escherichia coli. Intrinsic fluorescence, FRET, and Fourier transform infrared difference spectroscopy reveal that replacement of Lys-377 with either Cys, Val, Arg, or Asp disables both Na(+) and melibiose binding. On the other hand, molecular dynamics simulations extending up to 200-330 ns reveal that Lys-377 (helix XI) interacts with the anionic side chains of two of the three putative ligands for cation binding (Asp-55 and Asp-59 in helix II). When Asp-59 is protonated during the simulations, Lys-377 preferentially interacts with Asp-55. Interestingly, when a Na(+) ion is positioned in the Asp-55-Asp-59 environment, Asp-124 in helix IV (a residue essential for melibiose binding) reorients and approximates the Asp-55-Asp-59 pair, and all three acidic side chains act as Na(+) ligands. Under these conditions, the side chain of Lys-377 interacts with the carboxylic moiety of these three Asp residues. These data highlight the crucial role of the Lys-377 residue in the spatial organization of the Na(+) binding site. Finally, the analysis of the second-site revertants of K377C reveals that mutation of Ile-22 (in helix I) preserves Na(+) binding, whereas that of melibiose is largely abolished according to spectroscopic measurements. This amino acid is located in the border of the sugar-binding site and might participate in sugar binding through apolar interactions.

Crystallization of the carboxy-terminal region of the bacteriophage T4 proximal long tail fibre protein gp34.

The phage-proximal part of the long tail fibres of bacteriophage T4 consists of a trimer of the 1289 amino-acid gene product 34 (gp34). Different carboxy-terminal parts of gp34 have been produced and crystallized. Crystals of gp34(726-1289) diffracting X-rays to 2.9 Šresolution, crystals of gp34(781-1289) diffracting to 1.9 Šresolution and crystals of gp34(894-1289) diffracting to 3.0 and 2.0 Šresolution and belonging to different crystal forms were obtained. Native data were collected for gp34(726-1289) and gp34(894-1289), while single-wavelength anomalous diffraction data were collected for selenomethionine-containing gp34(781-1289) and gp34(894-1289). For the latter, high-quality anomalous signal was obtained.

The substitution of Arg149 with Cys fixes the melibiose transporter in an inward-open conformation.

The melibiose transporter from Escherichia coli (MelB) can use the electrochemical energy of either H(+), Na(+) or Li(+) to transport the disaccharide melibiose to the cell interior. By using spectroscopic and biochemical methods, we have analyzed the role of Arg149 by mutagenesis. According to Fourier transform infrared difference and fluorescence spectroscopy studies, R149C, R149Q and R149K all bind substrates in proteoliposomes, where the protein is disposed inside-out. Analysis of right-side-out (RSO) and inside-out (ISO) membrane vesicles showed that the functionally active R149Q and R149K mutants could bind externally added fluorescent sugar analog in both types of vesicles. In contrast, the non-transporting R149C mutant does bind the fluorescent sugar analog as well as melibiose and Na(+) in ISO, but not in RSO vesicles. Therefore, the mutation of Arg149 into cysteine restrains the orientation of transporter to an inward-open conformation, with the inherent consequences of a) reducing the frequency of access of outer substrates to the binding sites, and b) impairing active transport. It is concluded that Arg149, most likely located in the inner (cytoplasmic) half of transmembrane helix 5, is critically involved in the reorientation mechanism of the substrate-binding site accessibility in MelB.

Structural insights into the activation mechanism of melibiose permease by sodium binding.

The melibiose carrier from Escherichia coli (MelB) couples the accumulation of the disaccharide melibiose to the downhill entry of H(+), Na(+), or Li(+). In this work, substrate-induced FTIR difference spectroscopy was used in combination with fluorescence spectroscopy to quantitatively compare the conformational properties of MelB mutants, implicated previously in sodium binding, with those of a fully functional Cys-less MelB permease. The results first suggest that Asp55 and Asp59 are essential ligands for Na(+) binding. Secondly, though Asp124 is not essential for Na(+) binding, this acidic residue may play a critical role, possibly by its interaction with the bound cation, in the full Na(+)-induced conformational changes required for efficient coupling between the ion- and sugar-binding sites; this residue may also be a sugar ligand. Thirdly, Asp19 does not participate in Na(+) binding but it is a melibiose ligand. The location of these residues in two independent threading models of MelB is consistent with their proposed role.

In-plane and out-of-plane infrared difference spectroscopy unravels tilting of helices and structural changes in a membrane protein upon substrate binding.

Attenuated total reflection infrared (ATR-IR) difference spectroscopy stands out because of its ability to provide information on the interaction of substrates with membrane proteins in their native lipid bilayer environment. We show how the study and interpretation of the structural changes in membrane proteins upon substrate binding is simplified by obtaining ATR-IR difference spectra with polarized light and then computing the difference spectra in the z and x,y directions, where structural and orientation changes give specific difference absorbance patterns. In combination with a maximum-entropy band-narrowing method and some simple spectroscopic rules, the present approach allows us to unambiguously identify changes in the tilt of some helices in the secondary transporter melibiose permease following melibiose binding in the presence of sodium, suggesting the formation of an occluded state during the transport mechanism of the substrates.

Adenine nucleotide translocase 3 (ANT3) overexpression induces apoptosis in cultured cells.

Mitochondrial adenine nucleotide translocase 1 (ANT1), but not ANT2, can dominantly induce apoptosis. Nothing is known, however, about the apoptotic activity of ANT3. We have transfected HeLa cells with the three human ANT isoforms to compare their potential as inducers of apoptosis. Transient overexpression of ANT3 resulted, like ANT1, in apoptosis as shown by an increase in the sub-G1 fraction, annexin V staining, low DeltaPsi(m), and activation of caspases 9 and 3. Moreover, the apoptosis produced by ANT3 was inhibited by bongkrekic acid and by cyclosporin A. The pro-apoptotic activities of the ANT1 and ANT3 isoforms contrast with the lack of apoptotic activity of ANT2. This finding may help to identify the specific factors associated with the pro-apoptotic activities of ANT isoforms.