Initial synthesis and characterization of an immobilized heat shock protein 90 column for online determination of binding affinities
Anal Biochem. 2008 Feb 15;373(2):313-21. Epub 2007 Nov 6
Marszałł MP, Moaddel R, Jozwiak K, Bernier M, Wainer IW
Heat shock protein 90 alpha (Hsp90alpha) was immobilized on aminopropyl silica via the N terminus to create the Hsp90alpha(NT) column or via the C terminus to create the Hsp90alpha(CT) column. Binding to the exposed C terminus on the Hsp90alpha(NT) column was characterized using frontal chromatography and the C-terminus ligands coumermycin A(1) (CA1) and novobiocin (NOVO). The calculated K(d) values were 220+/-110 nM (CA1) and 100+/-20 nM (NOVO). Nonlinear chromatography was used to determine the association and dissociation rate constants associated with the NOVO-Hsp90alpha complex: 22.2+/-8.8 microM(-1) s(-1) and 2.7+/-0.6s(-1), respectively. Binding to the exposed N terminus on the Hsp90alpha(CT) column was characterized using frontal chromatography. The K(d) values of the N-terminus ligands geldanamycin (GM, 90+/-50 nM), 17-allylamino-17-demethoxygeldanamycin (17-AAG, 210+/-50 nM), and radicicol (RAD, 20+/-9 nM) were consistent with previously reported values. The effect of the immobilization on ATPase activity was investigated through the determination of IC(50) values for inhibition of ATPase activity on the Hsp90alpha(CT) column. The IC(50) for GM was 2.80+/-0.18 microM, and the relative IC(50) values were 17-AAG>GM>RAD, in agreement with previously reported values and indicating that immobilization had not affected ATPase activity or sensitivity to inhibition.
Molecular models of the interface between anterior pharynx-defective protein 1 (APH-1) and presenilin involving GxxxG motifs
ChemMedChem. 2008 Apr;3(4):627-34
Jozwiak K, Krzysko KA, Bojarski L, Gacia M, Filipek S
Gamma-secretase is an integral membrane protease, which is a complex of four membrane proteins. Improper functioning of gamma-secretase was found to be critical in the pathogenesis of Alzheimer's disease. Despite numerous efforts, the structure of the protease as well as its proteolytic mechanism remains poorly understood. In this work we constructed a model of interactions between two proteins forming gamma-secretase: APH-1 and presenilin. This interface is based on a highly conserved GxxxGxxxG motif in the APH-1 protein. It can form a tight contact with a small-residue AxxxAxxxG motif in presenilin. Here, four binding modes based on similar structures involving GxxxG motifs in glycophorin and aquaporin were proposed and verified. The resulting best model employs antiparallel orientations of interacting helices and is in agreement with the currently accepted topology of both proteins. This model can be used for further structural characterization of gamma-secretase and its components.
Exploring enantiospecific ligand-protein interactions using cellular membrane affinity chromatography: chiral recognition as a dynamic process
J Chromatogr B Analyt Technol Biomed Life Sci. 2008 Nov 1;875(1):200-7. doi: 10.1016/j.jchromb.2008.07.048
Jozwiak K, Moaddel R, Ravichandran S, Plazinska A, Kozak J, Patel S, Yamaguchi R, Wainer IW
The chiral recognition mechanisms responsible for the enantioselective binding on the alpha3beta4 nicotinic acetylcholine receptor (alpha3 beta4 nAChR) and human organic cation transporter 1 (hOCT1) have been reviewed. The results indicate that chiral recognition on the alpha3beta4 nAChR is a process involving initial tethering of dextromethorphan and levomethorphan at hydrophobic pockets within the central lumen followed by hydrogen bonding interactions favoring dextromethorphan. The second step is the defining enantioselective step. Studies with the hOCT1 indentified four binding sites within the transporter that participated in chiral recognition. Each of the enantiomers of the compounds used in the study interacted with three of these sites, while (R)-verapamil interacted with all four. Chiral recognition arose from the conformational adjustments required to produce optimum interactions. With respect to the prevailing interaction-based models, the data suggest that chiral recognition is a dynamic process and that the static point-based models should be amended to reflect this.
Identifying the binding site(s) for antidepressants on the Torpedo nicotinic acetylcholine receptor: [3H]2-azidoimipramine photolabeling and molecular dynamics studies
Biochim Biophys Acta. 2008 Dec;1778(12):2690-9. doi: 10.1016/j.bbamem.2008.08.019. Epub 2008 Sep 10
Sanghvi M, Hamouda AK, Jozwiak K, Blanton MP, Trudell JR, Arias HR
Radioligand binding, photoaffinity labeling, and docking and molecular dynamics were used to characterize the tricyclic antidepressant (TCA) binding sites in the nicotinic acetylcholine receptor (nAChR). Competition experiments indicate that the noncompetitive antagonist phencyclidine (PCP) inhibits [3H]imipramine binding to resting (closed) and desensitized nAChRs. [3H]2-azidoimipramine photoincorporates into each subunit from the desensitized nAChR with approximately 25% of the labeling specifically inhibited by TCP (a PCP analog), whereas no TCP-inhibitable labeling was observed in the resting (closed) state. For the desensitized nAChR and within the alpha subunit, the majority of specific [3H]2-azidoimipramine labeling mapped to a approximately 20 kDa Staphylococcus aureus V8 protease fragment (alphaV8-20; Ser173-Glu338). To further map the labeling site, the alphaV8-20 fragment was further digested with endoproteinase Lys-C and resolved by Tricine SDS-PAGE. The principal labeled fragment (11 kDa) was further purified by rpHPLC and subjected to N-terminal sequencing. Based on the amino terminus (alphaMet243) and apparent molecular weight, the 11 kDa fragment contains the channel lining M2 segment. Finally, docking and molecular dynamics results indicate that imipramine and PCP interact preferably with the M2 transmembrane segments in the middle of the ion channel. Collectively, these results are consistent with a model where PCP and TCA bind to overlapping sites within the lumen of the Torpedo nAChR ion channel.