Nucleotide Binding to the Multidrug Resistance P-Glycoprotein as Studied by ESR Spectroscopy

Abstract

Electron spin resonance (ESR) spectroscopy using spin-labeled ATP was used to study nucleotide binding to and structural transitions within the multidrug resistance P-glycoprotein, P-gp. Spin-labeled ATP (SL-ATP) with the spin label attached to the ribose, was observed to be an excellent substrate analogue for P-gp. SL-ATP was hydrolyzed in a drug-stimulated fashion at about 14% of the rate for normal ATP and allowed reversible trapping of the enzyme in transition and ground states. Equilibrium binding of a total of two nucleotides per P-gp was observed with a binding affinity of 366 microM in the presence of Mg2+ but in the absence of transport substrates such as verapamil. Binding of SL-ATP to wild-type P-gp in the presence of verapamil resulted in reduction of the protein-bound spin-label moiety, most likely due to a conformational transition within P-gp that positioned cysteines in close proximity to the spin label to allow chemical reduction of the radical. We circumvented this problem by using a mutant of P-gp in which all naturally occurring cysteines were substituted for alanines. Equilibrium binding of SL-ATP to this mutant P-gp resulted in maximum binding of two nucleotides; the binding affinity was 223 microM in the absence and 180 microM in the presence of verapamil. The corresponding ESR spectra of wild-type and Cys-less P-gp in the presence of SL-ATP indicate that a cysteine side chain of P-gp is located close to the ribose of the bound nucleotide. Trapping SL-ATP as an AlF(x)-adduct resulted in ESR spectra that showed strong immobilization of the radical, supporting the formation of a closed conformation of P-gp in its transition state. This study is the first to employ ESR spectroscopy with the use of spin-labeled nucleotide analogues to study P-glycoprotein. The study shows that SL-ATP is an excellent substrate analogue that will allow further exploration of structure and dynamics within the nucleotide binding domains of this important enzyme.

Nucleotide-induced Structural Changes in P-glycoprotein Observed by Electron Microscopy

“…Unfortunately as of now no high-resolution structure is available for a nucleotide-free ABC transporter with homology to Pgp (such as Sav1866). However, the nucleotide-free structure of ModBC shows the NBDs in quite a different conformation compared with ADP-bound Sav1866 (see below), consistent with the idea that the NBDs are undergoing large structural changes between nucleotide-free and nucleotide-bound forms (32).…”

“…In addition, orthovanadate was included in conjunction with MgATP or MgADP to produce the vanadate-trapped state (30). To grow crystals of nucleotide-bound Pgp, we initially tried to co-crystallize protein and nucleotide, but this approach did not result in ordered arrays, possibly due to heterogeneity in nucleotide occupancy as a consequence from the relatively modest affinity of Pgp for nucleotides (31,32). Crystallization was therefore started in the absence of nucleotide and once crystals were formed, nucleotide was added for various amounts of time before preparation of the crystals for electron microscopy (supplemental Fig.…”

In Silico Screening for Inhibitors of P-Glycoprotein That Target the Nucleotide Binding Domains

“…A well-established activity assay for ATP hydrolyzing enzymes (Delannoy et al, 2005;Hoffman et al, 2010) was used that couples the hydrolysis of ATP by P-gp to the oxidation of NADH to NAD 1 through the reactions of pyruvate kinase and lactate dehydrogenase (Vogel and Steinhart, 1976). These reactions were adapted to medium-throughput conditions on 96-well plates.…”

“…This cysteine-less mouse P-gp was used for assaying the potential inhibitor compounds. Purification of the protein was performed essentially as described by Delannoy et al (2005) with small modifications resulting in highly enriched P-gp in mixed micelles containing dodecyl maltoside and lysophosphatidyl choline. To obtain concentrated P-gp, the elution fraction of the Ni-NTA column was concentrated 70-to 100-fold using Amicon Ultra centrifugal filters (Amicon/Millipore Corp., Billerica, MA) with a molecular mass cutoff of 100,000 Da.…”

Human P-glycoprotein Contains a Greasy Ball-and-Socket Joint at the Second Transmission Interface

“…In this "nucleotide-sandwich conforma-tion," each nucleotide would interact with the phosphate-binding loop (P-loop) of one NBD and the ABC signature motif (LSGGQ) of the other NBD. ATP hydrolysis occurs by an alternating site mechanism as inhibition of ATPase activity at one site causes a complete loss of ATPase activity (12)(13)(14)(15).…”