Interactions and cooperativity between P-glycoprotein structural domains determined by thermal unfolding provides insights into its solution structure and function

Abstract

Structural changes in mouse P-glycoprotein (Pgp) induced by thermal unfolding were studied by differential scanning calorimetry (DSC), circular dichroism and fluorescence spectroscopy to gain insight into the solution conformation(s) of this ABC transporter that may not be apparent from current crystal structures. DSC of reconstituted Pgp showed two thermal unfolding transitions in the absence of MgATP, suggesting that each transition involved the cooperative unfolding of two or more interacting structural domains. A low calorimetric unfolding enthalpy and minimal structural changes were observed, which are hallmarks of the thermal unfolding of α-helical membrane proteins, because generally only the extramembranous regions undergo significant unfolding. Nucleotide binding increased the unfolding temperature of both transitions to the same extent, suggesting that one nucleotide binding domain (NBD) unfolds with each transition. Combined with the results from the two isolated NBDs, we propose that each DSC transition represents the cooperative unfolding of one NBD and the two contacting intracellular loops. Further, the presence of two transitions in both apo and MgATP bound wild-type Pgp suggests the NBD-dimeric conformation is transient, and that Pgp resides predominantly in the crystallographically observed inward-facing conformation with NBDs separated, even under conditions supporting continuous MgATP hydrolysis. In contrast, DSC of the vanadate-trapped MgADP·Pgp complex and the MgATP-bound catalytically inactive mutant, E552A/E1197A, show an additional transition at much higher temperature, corresponding to the unfolding of the nucleotide-trapped NBD-dimeric outward-facing conformation. The collective results indicate a strong preference for an NBD dissociated, inward-facing conformation of Pgp.

Replacing the eleven native tryptophans by directed evolution produces an active P-glycoprotein with site-specific, non-conservative substitutions

“…The far-UV CD spectrum of detergent-solubilized Pgp exhibited minima at 208 and 222 nm, as previously reported, and is typical for an α-helical protein (Fig. 6C) 84,85 . The shape of the far-UV CD spectra of W(3Cyto) and WL-Pgp was indistinguishable from Wt samples, suggesting that secondary and, in all likelihood, tertiary structure was maintained in the Trp mutants.…”

“…A second transition followed immediately after a sharp increase in the PMT voltage profile (gray circles, right axis). The increase in voltage suggested sample aggregation 85 , and coincided with a visible precipitation in the sample cuvette after heating above 50 °C. A comparison of the thermal unfolding profiles showed that the first transition was shifted to slightly lower temperatures in the W(3Cyto) (36.8 ± 0.5 °C), and significantly lower in the WL-Pgp (33.5 ± 0.3 °C).…”

Structural stability of purified human CFTR is systematically improved by mutations in nucleotide binding domain 1

“…A similar +5 °C shift in T m Trp due to H1402S was consistently observed in ΔRI/2PT/H1402S, ΔRI/2PT/M470V/H1402S, and 6SS/H1402S, suggesting that the shift in conformational population enhanced structural stability of the protein. Indeed, a similar stabilizing effect was observed in P-glycoprotein with equivalent mutations that promoted the occluded NBD dimer conformation with trapped MgATP [72,73]. A large body of literature reports on conformational stabilization due to specific mutations and combinations of mutations in G-protein coupled receptors (GPCRs).…”

“…In those cases, conformational stabilization was essential to obtain crystal structures in different states [74], [75], [76], [77]. Thermal unfolding studies with P-glycoprotein and other membrane proteins have shown that unfolding of α-helical transmembrane domains is unlikely to be detected by Trp fluorescence over the temperature range of the present study [55,73,78,79]. In CFTR, 11 of the 23 intrinsic Trp residues are situated in transmembrane domains.…”

Conformational dynamics of P-glycoprotein in lipid nanodiscs and detergent micelles reveal complex motions on a wide time scale

“…Most models for P-gp dynamics include intimate contact between NBD1 and NBD2 upon formation of the OF state, whereas they are well separated and apparently more solvent exposed in the IF state. In fact the NBDs thermodynamically stabilize one another in the vanadate-trapped state (29), which implies substantial inter-NBD interaction, as suggested by the effect of vanadate trapping on EX2 kinetics for many peptides spanning the NBDs. Thus, the combined H/DX results indicate that, either the expected contact between the NBDs upon vanadate trapping of the OF state only modestly affects solvent access to most structural elements including those that are stabilized by NBD dimer formation, or the NBDs remain in a dynamic equilibrium between states that allow solvent access even when the ATPase activity is persistently inhibited and the NBDs form a dimer.…”