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Multidrug efflux pumps, such as P-glycoprotein (ABCB1), present major barriers to the success of chemotherapy in a number of clinical settings. Molecular details of the multidrug efflux process by ABCB1 remain elusive, in particular, the interdomain communication associated with bioenergetic coupling. The present investigation has focused on the role of transmembrane helix 12 (TM12) in the multidrug efflux process of ABCB1. Cysteine residues were introduced at various positions within TM12, and their effect on ATPase activity, nucleotide binding, and drug interaction were assessed. Mutation of several residues within TM12 perturbed the maximal ATPase activity of ABCB1, and the underlying cause was a reduction in basal (i.e., drug-free) hydrolysis of the nucleotide. Two of the mutations (L976C and F978C) were found to reduce the binding of [gamma-(32)P]-azido-ATP to ABCB1. In contrast, the A980C mutation within TM12 enhanced the rate of ATP hydrolysis; once again, this was due to modified basal activity. Several residues also caused reductions in the potency of stimulation of ATP hydrolysis by nicardipine and vinblastine, although the effects were independent of changes in drug binding per se. Overall, the results indicate that TM12 plays a key role in the progression of the ATP hydrolytic cycle in ABCB1, even in the absence of the transported substrate.

The ATP binding cassette (ABC) transporters form one of the largest protein superfamilies. They use the energy of ATP hydrolysis to transport chemically diverse ligands across membranes. An alternating access mechanism in which the transporter switches between inward- and outward-facing conformations has been proposed to describe the translocation process. One of the main open questions in this process is the degree of opening of the transporter at different stages of the transport cycle, as crystal structures and biochemical data have suggested a wide range of distances between nucleotide binding domains. Recently, the crystal structure of McjD, an antibacterial peptide ABC transporter from Escherichia coli, revealed a new occluded intermediate state of the transport cycle. The transmembrane domain is closed on both sides of the membrane, forming a cavity that can accommodate its ligand, MccJ25, a lasso peptide of 21 amino acids. In this work, we investigate the degree of opening of the transmembrane cavity required for ligand translocation. By means of steered molecular dynamics simulations, the ligand was pulled from the internal cavity to the extracellular side. This resulted in an outward-facing state. Comparison with existing outward-facing crystal structures shows a smaller degree of opening in the simulations, suggesting that the large conformational changes in some crystal structures may not be necessary even for a large substrate like MccJ25.

Peptide-membrane interactions are important for understanding the binding, partitioning, and folding of membrane proteins; the activity of antimicrobial and fusion peptides; and a number of other processes. We describe molecular dynamics simulations (10-25 ns) of two pentapeptides Ace-WLXLL (with X = Arg or Lys side chain) (White, S. H., and Wimley, W.C. (1996) Nat. Struct. Biol. 3, 842-848) in water and three different membrane mimetic systems: (i) a water/cyclohexane interface, (ii) water-saturated octanol, and (iii) a solvated dioleoylphosphatidylcholine bilayer. A salt bridge is found between the protonated Arg or Lys side chains with the carboxyl terminus at the three interfaces. In water/cyclohexane, the salt bridge is most exposed to the water phase and least stable. In water/octanol and the lipid bilayer systems, the salt bridge once formed persists throughout the simulations. In the lipid bilayer, the salt bridge is more stable when the peptide penetrates deeper into the bilayer. In one of two peptides, a cation-pi interaction between the Arg and the Trp side chains is stable in the lipid bilayer for about 15 ns before breaking. In all cases, the conformations of the peptides are restricted by their presence at the interface and can be assigned to a few major conformational clusters. Side chains facing the water phase are most mobile. In the lipid bilayer, the peptides remain in the interface area, where they overlap with the carbonyl area of the lipid bilayer and perturb the local density profile of the bilayer. The tryptophan side chain remains in the water-lipid interface, where it interacts with the lipid choline group and forms hydrogen bonds with the ester carbonyl of the lipid and with water in the interface.

The transport of lipids between membrane leaflets, also known as flip-flop, is a key process in regulating the lipid composition of biological membranes. It is also important for the growth of biogenic membranes that are the site for lipid synthesis. It has been shown that the mere presence of transmembrane alpha-helical peptides or proteins enhances the rate lipid flip-flop [Kol et al. (2001) Biochemistry 40, 10500-10506]. Using computational models of natural phospholipids with different headgroups, we calculated the free energy profiles for transferring single phospholipids from bulk water to the center of a dioleylphosphatidylcholine (DOPC) bilayer in the presence of transmembrane helices. The free energy barrier for phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) flip-flop decreased by a few kilojoules per mole when a WALP23 or KALP23 peptide was present in the membrane, while the barrier for PC was not affected. We observed large bilayer deformations during lipid flip-flop when the hydrophilic headgroup is in the hydrophobic interior of the bilayer. The presence of KALP23 or WALP23 decreased the size and stability of these defects, suggesting integral membrane proteins affect the mechanism of flip-flop. There was a large decrease in the free energy of desorption for PE and PG when transmembrane peptides were present. This suggests specific PE and PG interactions with the peptide have a large affect on their stability in the membrane, with implications on cellular lipid and protein trafficking.

Acyl carrier protein (ACP) is the central player in fatty acid (FA) biosynthesis. It covalently binds all FA intermediates and presents them to the enzymes needed for elongation. Bacterial ACP must interact with a large number of proteins, which raises the question of how different acyl-ACPs are recognized and distinguished from each other. We performed molecular dynamics (MD) simulations of the FA synthase intermediates beta-ketoacyl-, beta-hydroxyacyl, and trans-2-enoyl-ACP spanning from 4 to 18 carbon groups in length. These forms of acyl-ACP have largely yet to be characterized experimentally, and our simulations provide a first insight into these structures. The simulations were conducted with the acyl chain directed into the solvent, as well as in a solvent-protected conformation inside the hydrophobic pocket of Escherichia coli ACP. Spontaneous migration from the solvent-exposed state into the hydrophobic binding pocket of ACP was seen in each of the intermediate classes studied, but not in all the individual simulations. This confirms that the intermediates can enter and utilize the same hydrophobic pockets as saturated acyl chains. In addition, a recurring, novel association of the acyl chains with loop I of ACP was observed that may be occupied transiently before entry into the hydrophobic pocket. The MD simulations of the acyl chains in a solvent-shielded state reveal that the polar functional group in the beta position of the beta-ketoacyl and beta-hydroxyacyl chains anchors these moieties at the cavity entrance, while the chains without a polar group in the beta position lack this additional anchoring atom. This leads to a binding mode in which the beta-ketoacyl and beta-hydroxyacyl chains are positioned further from the bottom of the pocket compared to the saturated and enoyl chains, particularly in short chain (C12) ACPs. These observations suggest a rationale for how different acyl-ACP intermediates may be distinguished by FA synthase enzymes.

Reduced intracellular drug accumulation due to the activity of the drug efflux pump ABC (B1) is a major mechanism in the resistance of cancer cells to chemotherapy. ABC (B1) is a poly specific transporter, and the molecular mechanism of its complex translocation process remains to be elucidated. To understand the process will require information on the regions involved in drug binding and those that couple this event to nucleotide hydrolysis. The present investigation focuses on the cytosolic region of transmembrane helix 6 (TM6), which has been widely attributed with a central role in the translocation process. A series of ABC (B1) isoforms containing a unique cysteine within TM6 was constructed and the resultant proteins purified and reconstituted. Accessibility of the cysteines to covalent modification by maleimide reagents was measured for the basal, ATP bound and vanadate trapped conformations of each isoform. Residues at the two extremes of the TM6 region examined (amino acids 344 to 360) were considerably more accessible than the central segment, the latter of which also failed to undergo significant conformational changes during the catalytic cycle. Covalent modification of the cytosolic segment of TM6 did, however, attenuate drug stimulation of ATP hydrolysis and demonstrates an important role for this segment in coupling drug binding to ATP hydrolysis during translocation.

Multidrug transporters such as P-glycoprotein require considerable inter-domain communication to couple energy utilization with substrate translocation. Elucidation of the regions or residues involved in these communication pathways is a key step in the eventual molecular description of multidrug transport. We used cysteine-scanning mutagenesis to probe the functional involvement of residues along the cytoplasmic half of transmembrane segment 6 (TM6) and its extension toward the nucleotide binding domain. The mutation of one residue (G346C) in this segment adversely affected drug transport in cells. Further investigation using purified protein revealed that the underlying biochemical effect was a reduction in basal ATP hydrolysis. This G346C mutation also affected the stimulation of ATPase activity in a drug dependent manner but had no effect on drug binding, ATP binding, or ADP release. Homology modeling of P-glycoprotein indicated that the G346C mutation caused a steric interaction between TM5 and TM6, thereby precluding a helical movement required to support ATP hydrolysis.

The twin-arginine translocase (Tat) system is used by many bacteria to translocate folded proteins across the cytoplasmic membrane. The TatA subunit is the predicted pore-forming subunit and has been shown to form a homo-oligomeric complex. Through accessibility experiments using the thiol-reactive reagents 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid and Nalpha-(3-maleimidylproprionyl)biocytin toward site-specific cysteine mutants in TatA, we show that the N-terminus of TatA is located in the cytoplasm rather than the previously assumed periplasm. We also confirm previous observations that the C-terminus has a dual topology. By treatment with the membrane uncoupler carbonyl cyanide-m-chlorophenyl hydrazone, we show that the topological state of the C-terminus is dependent on the membrane potential. These results suggest two architectures of TatA in the membrane: one with a single transmembrane helix and the other with two transmembrane helices. Molecular models of both topologies were used to develop and cartoon a homo-oligomeric complex as a channel with a diameter of approximately 50 A and suggest that the double transmembrane helix topology might be the building block for the translocation channel. Additionally, in vivo cross-linking experiments of Gly2Cys and Thr22Cys mutants showed that Gly2, at the beginning of transmembrane helix-1, is in close proximity with Gly2 of a neighboring TatA, as Cys2 cross-linked immediately upon the addition of copper phenanthroline. On the other hand, Cys22, at the other end of the transmembrane helix, took at least 10 min to cross-link, suggesting that a possible movement or reorientation is required to bring this residue into proximity with a neighboring TatA subunit.

BtuF is the periplasmic binding protein (PBP) in the vitamin B(12) uptake system in Escherichia coli where it is associated with the ABC transporter BtuCD. When the ligand binds, PBPs generally display large conformational changes, commonly termed the Venus flytrap mechanism. BtuF belongs to a group of PBPs that, on the basis of crystal structures, does not appear to display such behavior. Using 480 ns multicopy molecular dynamics simulations of apo and holo forms of the protein, we investigate the dynamics of BtuF. We find BtuF to be more flexible than previously assumed, displaying clear opening and closing motions which are more pronounced in the apo form. The protein behavior is compatible with a PBP functional model that postulates a closed conformation for the ligand-bound state, whereas the empty form fluctuates between open and closed conformations. Elastic network normal-mode analysis suggests that all BtuF-like PBPs are capable of similar opening and closing motions. It also makes the typical Venus flytrap domain motions a likely common means of how PBP-ABC transporter interaction could occur.

Simulated annealing was performed to model parallel dimers of alpha-helical transmembrane peptides with the sequence L(11)XL(12), predicting left-handed coiled coil geometry in all cases. Insertion of peptides containing threonine, asparagine, alanine, phenylalanine, and leucine in position 12 into realistic model membranes showed these structures were stable for 20 ns of molecular dynamics simulation time. Threonine could participate in intermolecular hydrogen bonds, but predominantly formed hydrogen bonds to the backbone of the helix it resided on. These hydrogen bonds, although infrequent, appeared to promote closer association of polyleucine helices. Asparagine participated in multiple, rapidly fluctuating intermolecular and intramolecular hydrogen bonds, and may have slightly destabilized optimum van der Waals packing in favor of optimum hydrogen bonding. Coordinated rotations of transmembrane helices about their axes were observed, indicating helices may rotate around one another during the folding of membrane proteins or other processes. These rotations were inhibited by phenylalanine, suggesting a role for bulky residues in modulating membrane protein dynamics.