Conformational changes of the histidine ATP-binding cassette transporter studied by double electron–electron resonance spectroscopy

Abstract

The conformational dynamics of the histidine ABC transporter HisQMP2 from Salmonella enterica serovar Typhimurium, reconstituted into liposomes, is studied by site-directed spin labeling and double electron-electron resonance spectroscopy in the absence of nucleotides, in the ATP-bound, and in the post-hydrolysis state. The results show that the inter-dimer distances as measured between the Q-loops of HisP2 in the intact transporter resemble those determined for the maltose transporter in all three states of the hydrolysis cycle. Only in the presence of liganded HisJ the closed conformation of the nucleotide binding sites is achieved revealing the transmembrane communication of the presence of substrate. Two conformational states can be distinguished for the periplasmic moiety of HisQMP2 as detected by differences in distributions of interspin distances between positions 86 and 96 or 104 and 197. The observed conformational changes are correlated to proposed open, semi-open and closed conformations of the nucleotide binding domains HisP2. Our results are in line with a rearrangement of transmembrane helices 4 and 4' of HisQM during the closed to the semi-open transition of HisP2 driven by the reorientation of the coupled helices 3a and 3b to occur upon hydrolysis.

ATP-dependent Conformational Changes Trigger Substrate Capture and Release by an ECF-type Biotin Transporter

“…In particular, the distance of the Q-loops in the MalK dimer of the maltose ABC importer MalFGK was demonstrated to decrease in the ATP-bound state and to increase upon ATP hydrolysis (50,52). A similar response was observed for the HisP dimer of the transporter for positively charged amino acids (HisQMP) (53). In order to test whether closure and reopening occurs in a similar way in ATPases of ECF transporters, we measured distances between the Q-helices of the BioM dimer that correspond to the Q-loops of canonical ABC importers (14).…”

“…Interestingly, the EPR data reflect three distinguishable conformations related to the three states of the hydrolysis cycle. Three different cycle-related conformations were also reported for the canonical type I ABC importers MalFGK and HisQMP, where the apo-state, the ADP-bound, and the ATP-bound states represent an open, a semiopen, and a closed conformation of the ATPase dimer, respectively (37,52,53,(61)(62)(63)(64)(65)(66)(67). In particular, DEER data obtained with the Ala-85/Ala-85Ј positions located in the Q-loops of the well characterized MalK dimer revealed distances between the two residues of 28, 18.5, and 19.5 Å for the apo-, ATP-bound, and post-hydrolytic state, respectively (52).…”

The Synergetic Effects of Combining Structural Biology and EPR Spectroscopy on Membrane Proteins

“…The mechanisms as proposed are a result of combining structural, functional, biophysical and spectroscopic studies. For type 1 and type 2 importers each one common transport model has been proposed, which are supported by several EPR studies [100][101][102][103][104][105][106][107][108][109][110]. First EPR studies on the dynamics of ECF transporters were presented recently [111,112], while the final mechanism is still under debate.…”

ATP–Binding Cassette Transporter Structure Changes Detected by Intramolecular Fluorescence Energy Transfer for High-Throughput Screening

“…Electron paramagnetic resonance and double electron-electron resonance spectroscopy have been used to probe the conformational dynamics of ABC transporters (Zou and McHaourab, 2010;Schultz et al, 2011a,b;Rice et al, 2013;Joseph et al, 2014;Mishra et al, 2014;Sippach et al, 2014 ). One study determined that the histidine ABC transporter sampled open, semi-open, and closed conformations (Sippach et al, 2014). The closed conformation of the NBDs was only achieved in the presence of the ligand.…”

“…Moreover, on the basis of the complexity of the transport process, the large range of substrate sizes, and the diversity of ABC proteins, it is probable that additional structural substates/intermediates exist besides the two conformations revealed by the available crystal structures (Kerr et al, 2010;Jin et al, 2012;Korkhov et al, 2012;Choudhury et al, 2014;Du et al, 2014;Srinivasan et al, 2014). Electron paramagnetic resonance and double electron-electron resonance spectroscopy have been used to probe the conformational dynamics of ABC transporters (Zou and McHaourab, 2010;Schultz et al, 2011a,b;Rice et al, 2013;Joseph et al, 2014;Mishra et al, 2014;Sippach et al, 2014 ). One study determined that the histidine ABC transporter sampled open, semi-open, and closed conformations (Sippach et al, 2014).…”