Dynamics of α-helical subdomain rotation in the intact maltose ATP-binding cassette transporter Cédric Orellea, Frances Joan D. Alvareza, Michael L. Oldhamb, Arnaud Orellea, Theodore E. Wileya, Jue Chenb, and Amy L. Davidsona,1 aChemistry Department, and bHoward Hughes Medical Institute, Purdue University, 560 Oval Dr, West Lafayette, IN 47906 Edited by Christopher Miller, Brandeis University, Waltham, MA, and approved October 4, 2010 (received for review May 12, 2010) ATP-binding cassette (ABC) transporters are powered by a nucleo- events, a ∼10° rotation of the α-helical subdomains relative to tide-binding domain dimer that opens and closes during cycles of the RecA-like subdomains and a ∼14° rotation of the RecA-like ATP hydrolysis. These domains consist of a RecA-like subdomain subdomains relative to the C-terminal regulatory domains (23). and an α-helical subdomain that is specific to the family. Many Analysis of the helical subdomain rotation is crucial for a detailed studies on isolated domains suggest that the helical subdomain ro- understanding of how ATP hydrolysis is stimulated by MBP and tates toward the RecA-like subdomain in response to ATP binding, coupled to transport. We therefore determined the requirements moving the family signature motif into a favorable position to in- for subdomain rotation and defined the sequence of rotational teract with the nucleotide across the dimer interface. Moreover, events in the catalytic cycle. the transmembrane domains are docked into a cleft at the interface between these subdomains, suggesting a putative role of the Results rotation in interdomain communication. Electron paramagnetic Positioning Spin Labels to Detect α-Helical Subdomain Rotation in resonance spectroscopy was used to study the dynamics of this MalK. The maltose transporter has been crystallized in an inward- Escherichia coli rotation in the intact maltose transporter MalFGK2. facing conformation in the absence of nucleotide (23), and an This importer requires a periplasmic maltose-binding protein (MBP) outward-facing conformational intermediate, stabilized by bind- that activates ATP hydrolysis by promoting the closure of the ing of both ATP and MBP, that may resemble the transition state cassette dimer (MalK2). Whereas this rotation occurred during the (22, 24, 25) (Fig. 1A). Alignment of the RecA-like subdomains of BIOCHEMISTRY transport cycle, it required not only trinucleotide, but also MBP, these two structures (Fig. 1B) reveals an inward rotation of the suggesting it is part of a global conformational change in the trans- helical subdomain toward the RecA-like subdomain, as seen in 2þ porter. Interaction of AMP-PNP-Mg and a MBP that is locked in many isolated NBD structures upon ATP binding (5–8). These a closed conformation induced a transition from open MalK2 to two subdomains are connected by the Q-loop (27) that lines part semiopen MalK2 without significant subdomain rotation. Inward of the cleft surrounding the coupling helix of the transmembrane rotation of the helical subdomain and complete closure of MalK2 domains MalF and MalG. The Q-loop is expected to undergo therefore appear to be coupled to the reorientation of transmem- conformational changes upon subdomain rotation that might brane helices and the opening of MBP, events that promote trans- be detected as a change in mobility of an attached spin label fer of maltose into the transporter. After ATP hydrolysis, the helical (SL). Residues 83–87 in the Q-loop were individually mutated subdomain rotates out as MalK2 opens, resetting the transporter in to cysteine. Residues in the helical subdomain (residues V120, an inward-facing conformation. Q122, and A124) and in the RecA-line subdomain (residues A167, L168, and Q171) (Fig. 1B) were also mutated to cysteines, EPR spectroscopy ∣ transport mechanism ∣ membrane protein individually and in pairs, to measure changes in the distance between these two subdomains upon rotation. Residues were TP-binding cassette (ABC) transporters belong to one of the selected for mutation based on distance constraints (28, 29) Alargest protein superfamilies in organisms, and mediate the and side-chain solvent-accessibility [http://mobyle.rpbs.univ- translocation of a wide range of substrates across the membrane paris-diderot.fr/cgi-bin/portal.py?form=ASA (30)], to maximize (1). These transporters typically contain two transmembrane do- spin-labeling efficiency. mains (TMDs) and are energized by a nucleotide-binding domain Plasmids carrying mutation(s) encoding the cysteine substitu- (NBD) dimer that closes and opens during cycles of ATP binding tions in the malK gene were used to transform an E. coli strain and hydrolysis (2, 3). Each NBD consists of a RecA-like subdo- containing a chromosomal deletion of malK, and were tested main, found in numerous ATPases (4), and an α-helical subdo- for function by complementation, as judged by the red color main that is specific to the ABC family (5). Crystallographic of the colonies on maltose MacConkey agar plates (22). By this studies (5–8) and molecular dynamic simulations (9, 10), criterion, all of the substitutions in the helical and RecA-like sub- performed on isolated NBDs, suggest that the helical subdomain rotates toward the RecA-like subdomain in response to ATP domains were functional, but only one of the Q-loop substitutions binding. This rotation positions the ABC family signature motif (S83C) was functional. The mutants were purified, spin-labeled, to interact with nucleotide across the dimer interface so that the and tested for MBP-stimulated ATPase activity (Table S1). All NBDs can close to hydrolyze ATP. After ATP hydrolysis, it is of the double mutants showed a strong stimulation by MBP,which suggested that the helical subdomain rotates away (5, 11). In is characteristic of the maltose transporter (21). However, only addition, the TMDs contact the NBDs at the interface between four of the double mutants, those that retained reasonably high these subdomains (12–15), suggesting a putative role of the rota- tion in interdomain communication (16). Here, we used site- Author contributions: C.O. and A.L.D. designed research; C.O., F.J.D.A., M.L.O., A.O., and directed spin labeling electron paramagnetic resonance (EPR) T.E.W. performed research; F.J.D.A., M.L.O., and J.C. contributed new reagents/analytic spectroscopy (17, 18) to study the dynamics of this rotation in tools; C.O., J.C., and A.L.D. analyzed data; and C.O. and A.L.D. wrote the paper. the intact Escherichia coli maltose transporter MalFGK2 (19, 20). The authors declare no conflict of interest. A periplasmic maltose-binding protein (MBP) delivers the sub- This article is a PNAS Direct Submission. strate to the transporter and stimulates its ATPase activity (21). 1To whom correspondence should be addressed. E-mail: [email protected]. In the maltose transporter, closure of the NBD dimer requires This article contains supporting information online at www.pnas.org/lookup/suppl/ both ATP and MBP (22) and involves two separate rotational doi:10.1073/pnas.1006544107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1006544107 PNAS Early Edition ∣ 1of6 Downloaded by guest on September 27, 2021 (ADP-Mg2þ-bound) state (22), increased the mobility of S83C- SL relative to the catalytic intermediate, though the spectrum was not superimposable with that of the apo state (Fig. 2). Changes in mobility of SL positioned at V120C and Q122C in the helical subdomain were also seen only following the addition of both AMP-PNP-Mg2þ and maltose-MBP (Fig. 2). In contrast, changes in mobility of SL positioned at L168C and, to a lesser extent, Q171C in the RecA-like subdomain (Fig. S1) are seen upon ad- dition of AMP-PNP-Mg2þ only, in addition to the more substan- tial changes apparent when both ligands are present. Crystal structures of isolated NBDs indicate that when the NBDs are open, nucleotide binds to the RecA-like subdomain rather than the helical subdomain (31) consistent with the observation that Fig. 1. Structural views of the maltose ABC transporter. (A) Structures of the SLs positioned in the RecA-like subdomain, but not the helical maltose transporter in apo (Left, PDB 3FH6) and catalytic intermediate (Right, subdomain, sensed nucleotide binding. The altered mobilities of PDB 2R6G) states. The transmembrane domains MalF and MalG are both SLs in the helical subdomain in the catalytic intermediate may shown in orange. MBP is in blue. The RecA-like subdomain (gray), α-helical reflect helical subdomain rotation, which brings the SL side subdomain (red), and regulatory domain (purple) of MalK are distinguished. chains closer to residues in the RecA-like subdomain. ATP (green) and maltose (cyan) molecules are present only in the catalytic intermediate. (B) Closeup of the MalK/MalG interface. Structures of the apo and catalytic intermediate state are superimposed based on the RecA- EPR Analysis of Doubly Labeled Mutants in the Apo State. To demon- like subdomain to visualize the helical subdomain rotation and movement strate the rotation of the helical subdomain relative to the RecA- of the coupling helix. MalG is rendered in yellow (apo state) and orange (cat- like subdomain, we tested several different pairs of spin-labels to alytic intermediate state). The coupling helix (or EAA loop) of MalG is shown determine the distance between these subdomains (Table S2). as a cylinder. The RecA-like and helical subdomains of MalK (colored as in A) When the interspin distance is >25 Å, the spectrum of a doubly from the apo and catalytic intermediate states are shown in lighter and dar- labeled protein in continuous wave (cw)-EPR strictly corresponds ker hues, respectively (figure modified from ref. 23). Residue Q82 defining to the sum of the spectra of the noninteracting single mutants. the Q-loop (blue) is shown as stick for the catalytic intermediate state. Interspin distances can be determined at room temperature in Spheres indicate the positions of residues spin-labeled in this study (Q122 – and A167 are indicated).