32 research outputs found
Deletion of NH2− and COOH-terminal sequences destroys function of the Ca2+ ATPase of rabbit fast-twitch skeletal muscle sarcoplasmic reticulum
AbstractDeletion mutants of the Ca2+ ATPase of rabbit fast-twitch skeletal muscle sarcoplasmic reticulum (SERCA1a) were constructed and expressed in COS-1 cells. The mutants were expressed at levels 7- to 15-fold lower than the wild-type and were inactive. In vitro transcription-translation-insertion experiments showed that deletion of transmembrane sequences M1 and M2, but not of M8, M9, M10 or the NH2−terminal 30 amino acids inhibited the stable insertion of the enzyme into the membrane. Thus there was no correlation between loss of function and membrane insertion. A signal sequence for membrane insertion may exist in M1 and M2
Predicting P-Glycoprotein-Mediated Drug Transport Based On Support Vector Machine and Three-Dimensional Crystal Structure of P-glycoprotein
Human P-glycoprotein (P-gp) is an ATP-binding cassette multidrug transporter that confers resistance to a wide range of chemotherapeutic agents in cancer cells by active efflux of the drugs from cells. P-gp also plays a key role in limiting oral absorption and brain penetration and in facilitating biliary and renal elimination of structurally diverse drugs. Thus, identification of drugs or new molecular entities to be P-gp substrates is of vital importance for predicting the pharmacokinetics, efficacy, safety, or tissue levels of drugs or drug candidates. At present, publicly available, reliable in silico models predicting P-gp substrates are scarce. In this study, a support vector machine (SVM) method was developed to predict P-gp substrates and P-gp-substrate interactions, based on a training data set of 197 known P-gp substrates and non-substrates collected from the literature. We showed that the SVM method had a prediction accuracy of approximately 80% on an independent external validation data set of 32 compounds. A homology model of human P-gp based on the X-ray structure of mouse P-gp as a template has been constructed. We showed that molecular docking to the P-gp structures successfully predicted the geometry of P-gp-ligand complexes. Our SVM prediction and the molecular docking methods have been integrated into a free web server (http://pgp.althotas.com), which allows the users to predict whether a given compound is a P-gp substrate and how it binds to and interacts with P-gp. Utilization of such a web server may prove valuable for both rational drug design and screening
P-glycoprotein ATPase activity requires lipids to activate a switch at the first transmission interface
AbstractP-glycoprotein (P-gp) is an ABC (ATP-Binding Cassette) drug pump. A common feature of ABC proteins is that they are organized into two wings. Each wing contains a transmembrane domain (TMD) and a nucleotide-binding domain (NBD). Drug substrates and ATP bind at the interface between the TMDs and NBDs, respectively. Drug transport involves ATP-dependent conformational changes between inward- (open, NBDs far apart) and outward-facing (closed, NBDs close together) conformations. P-gps crystallized in the presence of detergent show an open structure. Human P-gp is inactive in detergent but basal ATPase activity is restored upon addition of lipids. The lipids might cause closure of the wings to bring the NBDs close together to allow ATP hydrolysis. We show however, that cross-linking the wings together did not activate ATPase activity when lipids were absent suggesting that lipids may induce other structural changes required for ATPase activity. We then tested the effect of lipids on disulfide cross-linking of mutants at the first transmission interface between intracellular loop 4 (TMD2) and NBD1. Mutants L443C/S909C and L443C/R905C but not G471C/S909C and V472C/S909C were cross-linked with oxidant when in membranes. The mutants were then purified and cross-linked with or without lipids. Mutants G471C/S909C and V472C/S909C cross-linked only in the absence of lipids whereas mutants L443C/S909C and L443C/R905C were cross-linked only in the presence of lipids. The results suggest that lipids activate a switch at the first transmission interface and that the structure of P-gp is different in detergents and lipids
Drugs Modulate Interactions between the First Nucleotide-Binding Domain and the Fourth Cytoplasmic Loop of Human P‑Glycoprotein
Drug substrates stimulate
ATPase activity of the P-glycoprotein
(P-gp) ATP-binding cassette drug pump by an unknown mechanism. Cross-linking
analysis was performed to test if drug substrates stimulate P-gp ATPase
activity by altering cross-talk at the first transmission interface
linking the drug-binding [intracellular loop 4 (S909C)] and first
nucleotide-binding domains [NBD1 (V472C or L443C)]. In the absence
of lipid (inactive P-gp), only V472C/S909C showed cross-linking. Drugs
blocked V472C/S909C cross-linking. In the presence of lipids (active
P-gp), drug substrates promoted only L443C/S909C cross-linking. This
suggests that drug substrates stimulate ATPase activity through a
conformational change that shifts Ser909 away from Val472 and toward
Leu443
Drug Rescue Distinguishes between Different Structural Models of Human P‑Glycoprotein
There
is no high-resolution crystal structure of the human P-glycoprotein
(P-gp) drug pump. Homology models of human P-gp based on the crystal
structures of mouse or <i>Caenorhabditis elegans</i> P-gps
show large differences in the orientation of transmembrane segment
5 (TM5). TM5 is one of the most important transmembrane segments involved
in drug–substrate interactions. Drug rescue of P-gp processing
mutants containing an arginine at each position in TM5 was used to
identify positions facing the lipid or internal aqueous chamber. Only
the model based on the <i>C. elegans</i> P-gp structure
was compatible with the drug rescue results
A Salt Bridge in Intracellular Loop 2 Is Essential for Folding of Human P‑Glycoprotein
There is no high-resolution structure
of the human P-glycoprotein
(P-gp, ABCB1) drug pump. Homology models based on the crystal structures
of mouse and <i>Caenorhabditis elegans</i> P-gps show extensive
contacts between intracellular loop 2 (ICL2, in the first transmembrane
domain) and the second nucleotide-binding domain. Human P-gp modeled
on these P-gp structures yields different ICL2 structures. Only the
model based on the <i>C. elegans</i> P-gp structure predicts
the presence of a salt bridge. We show that the Glu256–Arg276
salt bridge was critical for P-gp folding