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Structural Dynamics of the Heterodimeric ABC Transporter TM287/288 Induced by ATP and Substrate Binding

TM287/288 is a heterodimeric ATP-binding cassette (ABC) transporter, which harnesses the energy of ATP binding and hydrolysis at the nucleotide-binding domains (NBDs) to transport a wide variety of molecules through the transmembrane domains (TMDs) by alternating inward- and outward-facing conformations. Here, we conducted multiple 100 ns molecular dynamics simulations of TM287/288 in different ATP- and substrate-bound states to elucidate the effects of ATP and substrate binding. As a result, the binding of two ATP molecules to the NBDs induced the formation of the consensus ATP-binding pocket (ABP2) or the NBD dimerization, whereas these processes did not occur in the presence of a single ATP molecule or when the protein was in its apo state. Moreover, binding of the substrate to the TMDs enhanced the formation of ABP2 through allosteric TMD–NBD communication. Furthermore, in the apo state, α-helical subdomains of the NBDs approached each other, acquiring a conformation with core half-pockets exposed to the solvent, appropriate for ATP binding. We propose a “core-exposed” model for this novel conformation found in the apo state of ABC transporters. These findings provide important insights into the structural dynamics of ABC transporters

ATP Hydrolysis Mechanism in a Maltose Transporter Explored by QM/MM Metadynamics Simulation

Translocation of substrates across the cell membrane by adenosine 5′-triphosphate (ATP)-binding cassette (ABC) transporters depends on the energy provided by ATP hydrolysis within the nucleotide-binding domains (NBDs). However, the detailed mechanism remains unclear. In this study, we focused on maltose transporter NBDs (MalK₂) and performed a quantum mechanical/molecular mechanical (QM/MM) well-tempered metadynamics simulation to address this issue. We explored the free-energy profile along an assigned collective variable. As a result, it was determined that the activation free energy is approximately 10.5 kcal/mol, and the reaction released approximately 3.8 kcal/mol of free energy, indicating that the reaction of interest is a one-step exothermic reaction. The dissociation of the ATP γ-phosphate seems to be the rate-limiting step, which supports the so-called dissociative model. Moreover, Glu159, located in the Walker B motif, acts as a base to abstract the proton from the lytic water, but is not the catalytic base, which corresponds to an atypical general base catalysis model. We also observed two interesting proton transfers: transfer from the His192 ε-position nitrogen to the dissociated inorganic phosphate, Pi, and transfer from the Lys42 side chain to adenosine 5′-diphosphate β-phosphate. These proton transfers would stabilize the posthydrolysis state. Our study provides significant insight into the ATP hydrolysis mechanism in MalK₂ from a dynamical viewpoint, and this insight would be applicable to other ABC transporters

Analysis of the Free Energy Landscapes for the Opening–Closing Dynamics of the Maltose Transporter ATPase MalK₂ Using Enhanced-Sampling Molecular Dynamics Simulation

Protein dynamics are considered significant for many physiological processes, such as metabolism, biomolecular recognition, and the regulation of several vital cellular processes. Due to their flexibility, proteins may stay in different substates with or without the existence of the cognate substrates. To describe these phenomena, two models have been proposed: the “induced fit” and the “conformational selection” mechanisms. In this study, we used MalK₂, the subunits that mainly include the nucleotide-binding domains (NBDs) of the maltose transporter from Escherichia coli, as a target to understand the NBD dimerization mechanism. Accelerated and conventional molecular dynamics have been performed. The results revealed that Mg–ATP binding to MalK₂ led to a significant change in the free energy profile and thus stabilized the closed conformation. On the contrary, when Mg–ATP was removed, the open conformation would be favored. The fact that ligand binding induces a drastic free energy change leads to a significant inference: MalK₂ dimerization would occur through the induced-fit mechanism rather than the conformational selection mechanism. This study sheds new light on the NBD dimerization mechanism and would be of wide applicability to other ABC transporters

ATP Hydrolysis Mechanism in a Maltose Transporter Explored by QM/MM Metadynamics Simulation

Translocation of substrates across the cell membrane by adenosine 5′-triphosphate (ATP)-binding cassette (ABC) transporters depends on the energy provided by ATP hydrolysis within the nucleotide-binding domains (NBDs). However, the detailed mechanism remains unclear. In this study, we focused on maltose transporter NBDs (MalK₂) and performed a quantum mechanical/molecular mechanical (QM/MM) well-tempered metadynamics simulation to address this issue. We explored the free-energy profile along an assigned collective variable. As a result, it was determined that the activation free energy is approximately 10.5 kcal/mol, and the reaction released approximately 3.8 kcal/mol of free energy, indicating that the reaction of interest is a one-step exothermic reaction. The dissociation of the ATP γ-phosphate seems to be the rate-limiting step, which supports the so-called dissociative model. Moreover, Glu159, located in the Walker B motif, acts as a base to abstract the proton from the lytic water, but is not the catalytic base, which corresponds to an atypical general base catalysis model. We also observed two interesting proton transfers: transfer from the His192 ε-position nitrogen to the dissociated inorganic phosphate, Pi, and transfer from the Lys42 side chain to adenosine 5′-diphosphate β-phosphate. These proton transfers would stabilize the posthydrolysis state. Our study provides significant insight into the ATP hydrolysis mechanism in MalK₂ from a dynamical viewpoint, and this insight would be applicable to other ABC transporters

Analysis of the Structural and Functional Roles of Coupling Helices in the ATP-Binding Cassette Transporter MsbA through Enzyme Assays and Molecular Dynamics Simulations

ATP-binding cassette (ABC) transporters are constructed from some common structural units: the highly conserved nucleotide-binding domains (NBDs), which work as a nucleotide-dependent engine for driving substrate transport, the diverse transmembrane domains (TMDs), which create the translocation pathway, and the coupling helices (CHs), which are located at the NBD–TMD interface. Although the CHs are believed to be essential for NBD–TMD communication, their roles remain unclear. In this study, we performed enzyme assays and molecular dynamics (MD) simulations of the ABC transporter MsbA and two MsbA mutants in which the amino acid residues of one of the CHs were mutated to alanines: (i) wild type (Wt), (ii) CH1 mutant (Mt1), and (iii) CH2 mutant (Mt2). The experiments show that the CH2 mutation decreases the ATPase activity (<i>k</i>_cat) compared with that of the Wt (a decrease of 32%), and a nearly equal degree of decrease in the ATP binding affinity (<i>K</i>_m) was observed for both Mt1 and Mt2. The MD simulations successfully accounted for several structural and dynamical origins for these experimental observations. In addition, on the basis of collective motion and morphing analyses, we propose that the reverse-rotational motions and noddinglike motions between the NBDs and TMDs are indispensable for the conformational transition between the inward- and outward-facing conformations. In particular, CH2 is significantly important for the occurrence of the noddinglike motion. These findings provide important insights into the structure–function relationship of ABC transporters

ATP-Induced Conformational Changes of Nucleotide-Binding Domains in an ABC Transporter. Importance of the Water-Mediated Entropic Force

ATP binding cassette (ABC) proteins belong to a superfamily of active transporters. Recent experimental and computational studies have shown that binding of ATP to the nucleotide binding domains (NBDs) of ABC proteins drives the dimerization of NBDs, which, in turn, causes large conformational changes within the transmembrane domains (TMDs). To elucidate the active substrate transport mechanism of ABC proteins, it is first necessary to understand how the NBD dimerization is driven by ATP binding. In this study, we selected MalKs (NBDs of a maltose transporter) as a representative NBD and calculated the free-energy change upon dimerization using molecular mechanics calculations combined with a statistical thermodynamic theory of liquids, as well as a method to calculate the translational, rotational, and vibrational entropy change. This combined method is applied to a large number of snapshot structures obtained from molecular dynamics simulations containing explicit water molecules. The results suggest that the NBD dimerization proceeds with a large gain of water entropy when ATP molecules bind to the NBDs. The energetic gain arising from direct NBD–NBD interactions is canceled by the dehydration penalty and the configurational-entropy loss. ATP hydrolysis induces a loss of the shape complementarity between the NBDs, which leads to the dissociation of the dimer, due to a decrease in the water-entropy gain and an increase in the configurational-entropy loss. This interpretation of the NBD dimerization mechanism in concert with ATP, especially focused on the water-mediated entropy force, is potentially applicable to a wide variety of the ABC transporters

Two-dimensional PMF surface of the motion of the ligand along the MFEP.

<p>(A) PMF is projected onto the MFEP images and onto the 1st PC of the <i>xyz</i>-coordinates of the mass center of the AMP adenine. The PMF is represented by the colored contour lines. (B) Tens of the MD snapshots are superimposed at (left image), and 4 snapshots shows the misbinding between the AMP ribose and Asp84 at (right image). The hydrogen bonds are indicated by the dashed yellow lines.</p

Committor tests characterizing the TSE.

<p>(A) The binned distributions of the final structures after 10 ns unrestrained MD simulations (blue bars), assigned by index of the nearest MFEP image (i.e., classified by the Voronoi tessellation). The MD simulations were executed from different initial distributions (red bars) at , 33, and 34. (B) The average structures of the MFEP images at (before the TSE), and 34 (after the TSE). The ligand and the residues of Thr31, Lys57, Arg88, Gly85, and Gln92 are represented by sticks. The hydrogen bonds are indicated by the dashed yellow lines.</p

Dehydration of an occluded water around the P-loop.

<p>The isosurface representation of the 3D distribution function of water oxygen (red) and hydrogen (white) around the P-loop at (A) , (B) 41, and (C) 42. The surfaces show the areas in which the atoms are distributed four times as probably as in the bulk phase. For comparison, the oxygens of crystal waters are shown for (D) the open (PDBid: 4ake) and (E) closed conformations (PDBid: 1ake). An occluded water molecule at and the corresponding crystal water of the open form are indicated by the circles. (F) Two-dimensional PMF surface as a function of the MFEP images and the distances of the LID-CORE domains. The PMF is represented by the colored contour lines. Regions of physical events (AMP-binding and dehydration) are encircled.</p