Mechanism for the transport of ammonia within carbamoyl phosphate synthetase determined by molecular dynamics simulations

Carbamoyl phosphate synthetase (CPS) is a member of the amidotransferase family of enzymes that uses the hydrolysis of glutamine as a localized source of ammonia for biosynthetic transformations. Molecular dynamics simulations for the transfer of ammonia and ammonium through a tunnel in the small subunit of CPS resulted in five successful trajectories for ammonia transfer, while ammonium was immobilized in a water pocket inside the small subunit of the heterodimeric protein. The observed molecular tunnel for ammonia transport is consistent with that suggested by earlier X-ray crystallography and site-directed mutation studies. His-353, Ser-47, and Lys-202, around the active site center in the small subunit, function cooperatively to deliver ammonia from the site of formation to the interface with the large subunit, via the exchange of hydrogen bonds with a critical water cluster within the tunnel. The NH 3 forms and breaks hydrogen bonds to Gly-292, Ser-35, Pro-358, Gly-293, and Thr-37 in a stepwise fashion "macroscopically" as it travels through the hydrophilic passage toward the subunit interface. The potential of mean force calculations along the ammonia transfer pathway indicates a low free-energy path for the translocation of ammonia with two barriers of 3.9 and 5.5 kcal/mol, respectively. These low free-energy barriers are consistent with the delivery of ammonia from the site of formation into a water reservoir toward the exit of the tunnel and migration through the hydrophilic leaving passage, respectively. The high overall free-energy barrier of 22.4 kcal/mol for the transport of ammonium additionally substantiates that the tunnel in the small subunit of CPS is not an ammonium but an ammonia channel.     

Intramembrane proton binding site linked to activation of bacterial pentameric ion channel

Prokaryotic orthologs of eukaryotic Cys-loop receptor channels recently emerged as structural and mechanistic surrogates to investigate this superfamily of intercellular signaling proteins. Here, we examine proton activation of the prokaryotic ortholog GLIC using patch clamp electrophysiology, mutagenesis, and molecular dynamics (MD) simulations. Whole-cell current recordings from human embryonic kidney (HEK) 293 cells expressing GLIC show half-maximal activation at pH 6, close to the pK(a) of histidine, implicating the three native His residues in proton sensing linked to activation. The mutation H235F abolishes proton activation, H277Y is without effect, and all nine mutations of His-127 prevent expression on the cell surface. In the GLIC crystal structure, His-235 on transmembrane (TM) α-helix 2, hydrogen bonds to the main chain carbonyl oxygen of Ile-259 on TM α-helix 3. MD simulations show that when His-235 is protonated, the hydrogen bond persists, and the channel remains in the open conformation, whereas when His-235 is deprotonated, the hydrogen bond dissociates, and the channel closes. Mutations of the proximal Tyr-263, which also links TM α-helices 2 and 3 via a hydrogen bond, alter proton sensitivity over a 1.5 pH unit range. MD simulations show that mutations of Tyr-263 alter the hydrogen bonding capacity of His-235. The overall findings show that His-235 in the TM region of GLIC is a novel proton binding site linked to channel activation.     

The cytoplasmic cage domain of the mechanosensitive channel MscS is a sensor of macromolecular crowding

Cells actively regulate the macromolecular excluded volume of the cytoplasm to maintain the reciprocal fraction of free aqueous solution that is optimal for intracellular processes. However, the mechanisms whereby cells sense this critical parameter remain unclear. The mechanosensitive channel of small conductance (MscS channel), which is the major regulator of turgor in bacteria, mediates efflux of small osmolytes in response to increased membrane tension. At moderate sustained tensions produced by a decrease in external osmolarity, MscS undergoes slow adaptive inactivation; however, it inactivates abruptly in the presence of cytoplasmic crowding agents. To understand the mechanism underlying this rapid inactivation, we combined extrapolated and equilibrium molecular dynamics simulations with electrophysiological analyses of MscS mutants to explore possible transitions of MscS and generated models of the resting and inactivated states. Our models suggest that the coupling of the gate formed by TM3 helices to the peripheral TM1–TM2 pairs depends on the axial position of the core TM3 barrel relative to the TM1–TM2 shaft and the state of the associated hollow cytoplasmic domain (“cage”). They also indicate that the tension-driven inactivation transition separates the gate from the peripheral helices and promotes kinks in TM3s at G113 and that this conformation is stabilized by association of the TM3b segment with the β domain of the cage. We found that mutations destabilizing the TM3b–β interactions preclude inactivation and make the channel insensitive to crowding agents and voltage; mutations that strengthen this association result in a stable closed state and silent inactivation. Steered simulations showed that pressure exerted on the cage bottom in the inactivated state reduces the volume of the cage in the cytoplasm and at the same time increases the footprint of the transmembrane domain in the membrane, implying coupled sensitivity to both membrane tension and crowding pressure. The cage, therefore, provides feedback on the increasing crowding that disengages the gate and prevents excessive draining and condensation of the cytoplasm. We discuss the structural mechanics of cells surrounded by an elastic cell wall where this MscS-specific feedback mechanism may be necessary.     

An ion gating mechanism of gastric H,K-ATPase based on molecular dynamics simulations

Gastric H,K-ATPase is an electroneutral transmembrane pump that moves protons from the cytoplasm of the parietal cell into the gastric lumen in exchange for potassium ions. The mechanism of transport against the established electrochemical gradients includes intermediate conformations in which the transferred ions are trapped (occluded) within the membrane domain of the pump. The pump cycle involves switching between the E1 and E2P states. Molecular dynamics simulations on homology models of the E2P and E1 states were performed to investigate the mechanism of K⁺ movement in this enzyme. We performed separate E2P simulations with one K⁺ in the luminal channel, one K⁺ ion in the occlusion site, two K⁺ ions in the occlusion site, and targeted molecular dynamics from E2P to E1 with two K⁺ ions in the occlusion site. The models were inserted into a lipid bilayer system and were stable over the time course of the simulations, and K⁺ ions in the channel moved to a consistent location near the center of the membrane domain, thus defining the occlusion site. The backbone carbonyl oxygen from residues 337 through 342 on the nonhelical turn of M4, as well as side-chain oxygen from E343, E795, and E820, participated in the ion occlusion. A single water molecule was stably bound between the two K⁺ ions in the occlusion site, providing an additional ligand and partial shielding the positive charges from one another. Targeted molecular dynamics was used to transform the protein from the E2P to the E1 state (two K⁺ ions to the cytoplasm). This simulation identified the separation of the water column in the entry channel as the likely gating mechanism on the luminal side. A hydrated exit channel also formed on the cytoplasmic side of the occlusion site during this simulation. Hence, water molecules became available to hydrate the ions. The movement of the M1M2 transmembrane segments, and the displacement of residues Q159, E160, Q110, and T152 during the conformational change, as well as the motions of E343 and L346, acted as the cytoplasmic-side gate.     

Size, motion, and function of the SecY translocon revealed by molecular dynamics simulations with virtual probes

We report a hybrid, coarse-grained and atomistic, molecular dynamics simulation study of the size, motion, and function of the SecY protein-conducting channel. Growing and pushing virtual soft ball constructs through the pore of SecY, we mimic the push-through of polypeptides, performed cotranslationally by the ribosome and posttranslationally by the SecA ATPase. Forced lateral opening of a "front gate" between transmembrane helices is also induced by the passage of the virtual probes, with implications for the membrane insertion of peptides. We conclude that the SecY channel can stretch to allow passage of peptides with transversal sizes of approximately 16 A. The observed motion of a transmembrane helical "plug" controlling the closed and open states of the channel is consistent with experimental results and confirms previous hypotheses. Additionally, the "hinge" region for front gate opening is observed to be highly mobile as postulated. Both the forced dilation of a "ring" of residues at the middle of the pore and the lateral opening of the front gate are shown to induce plug displacement, but neither accomplish a full-extent motion of the plug to the back of the channel. For probes whose passage does not destroy the resilience of the SecY, both lateral exit and full translocation are observed, despite the fact that applied forces were always in the direction along the pore axis. Lateral exit is accompanied by front gate opening and slight plug displacement, whereas full translocation is accompanied by large plug displacement but no apparent lateral opening. Simulations also reveal that dilating the pore ring is a more effective way to destabilize the plug than intercalation of a cylinder-like probe at the front gate. Based on the simulations, the existence of a family of diverse open states is proposed.     

State-dependent regulation of cystic fibrosis transmembrane conductance regulator (CFTR) gating by a high affinity Fe3+ bridge between the regulatory domain and cytoplasmic loop 3

The unique regulatory (R) domain differentiates the human CFTR channel from other ATP-binding cassette transporters and exerts multiple effects on channel function. However, the underlying mechanisms are unclear. Here, an intracellular high affinity (2.3 × 10(-19) M) Fe(3+) bridge is reported as a novel approach to regulating channel gating. It inhibited CFTR activity by primarily reducing an open probability and an opening rate, and inhibition was reversed by EDTA and phenanthroline. His-950, His-954, Cys-832, His-775, and Asp-836 were found essential for inhibition and phosphorylated Ser-768 may enhance Fe(3+) binding. More importantly, inhibition by Fe(3+) was state-dependent. Sensitivity to Fe(3+) was reduced when the channel was locked in an open state by AMP-PNP. Similarly, a K978C mutation from cytoplasmic loop 3 (CL3), which promotes ATP-independent channel opening, greatly weakened inhibition by Fe(3+) no matter whether NBD2 was present or not. Therefore, although ATP binding-induced dimerization of NBD1-NBD2 is required for channel gating, regulation of CFTR activity by Fe(3+) may involve an interaction between the R domain and CL3. These findings may support proximity of the R domain to the cytoplasmic loops. They also suggest that Fe(3+) homeostasis may play a critical role in regulating pathophysiological CFTR activity because dysregulation of this protein causes cystic fibrosis, secretary diarrhea, and infertility.     

pH-dependent channel gating in connexin26 hemichannels involves conformational changes in N-terminus

Connexin (Cx) hemichannels controlling an exchange of ions and metabolites between the cytoplasm and extracellular milieu can be modulated by the variation of intracellular pH during physiological and pathological conditions. To address the mechanism by which the pH exerts its effect on hemichannels, we have performed two 100-ns molecular dynamics simulations of the Cx26 channel in both acidic and neutral states. The results show that: 1) transmembrane domains undergo clockwise motions around the pore axis under both acidic and neutral conditions, while extracellular segments keep stable. 2) Under neutral condition, Cx26 has a tightly closed configuration that occurs through the assembly of N-terminal helix (NTH) region. This shows a constriction formed by the interhelical interactions of Asp2 and Met1 from neighboring NTH, which shapes the narrowest segment (pore radius<2?) of the pore, preventing the passage of ions from the extracellular side. This indicates that Asp2 may act as a channel gate. 3) Under the acidic condition, the constriction is relieved by the protonation of Asp2 causing interruption of interhelical interactions, Cx26 has a flexibly opening pore (pore radius>4.5?) around NTH region, allowing the passage of chloride ions unimpeded by the side-chain Asp2. While in the extracellular part two chloride ions interact with the side-chain Lys41 from three subunits. Finally, we provide a plausible mechanism of pH-dependent gating of hemichannel that involves protonation of the aspartic residues, suggesting that the pH sensitivity of hemichannel permeability is a sophisticated mechanism for cell regulating ion permeation.     

Influence of a lipid interface on protein dynamics in a fungal lipase.

Lipases catalyze lipolytic reactions and for optimal activity they require a lipid interface. To study the effect of a lipid aggregate on the behavior of the enzyme at the interfacial plane and how the aggregate influences an attached substrate or product molecule in time and space, we have performed molecular dynamics simulations. The simulations were performed over 1 to 2 ns using explicit SPC water. The interaction energies between protein and lipid are mainly due to van der Waals contributions reflecting the hydrophobic nature of the lipid molecules. Estimations of the protonation state of titratable residues indicated that the negative charge on the fatty acid is stabilized by interactions with the titratable residues Tyr-28, His-143, and His-257. In the presence of a lipid patch, the active site lid opens wider than observed in the corresponding simulations in an aqueous environment. In that lid conformation, the hydrophobic residues Ile-85, Ile-89, and Leu-92 are embedded in the lipid patch. The behavior of the substrate or product molecule is sensitive to the environment. Entering and leaving of substrate molecules could be observed in presence of the lipid patch, whereas the product forms strong hydrogen bonds with Ser-82, Ser-144, and Trp-88, suggesting that the formation of hydrogen bonds may be an important contribution to the mechanism by which product inhibition might take place.     

Identification of the active-site serine in human pancreatic lipase by chemical modification with tetrahydrolipstatin.

A chemical modification approach was used in this study to identify the active site serine residue of human pancreatic lipase. Purified human pancreatic lipase was covalently modified by incubation with [3H], [14C] tetrahydrolipstatin (THL), a potent inhibitor of pancreatic lipase. The radiolabeled lipase was digested with thermolysin, and the peptides were separated by HPLC. A single THL-peptide-adduct was obtained which was identical to that obtained earlier from porcine pancreatic lipase. This pentapeptide with the sequence VIGHS is covalently bound to a THL molecule via the side chain hydroxyl group of the serine unit corresponding to Ser-152 of the lipase. The selective cleavage of the THL-serine bond by mild acid treatment resulted in the formation of the delta-lactone Ro 40-4441 in high yield and clearly proves that THL is attached via an ester bond and with retention of stereochemistry at all chiral centers to the side chain hydroxyl group of Ser-152 of the lipase. The results obtained for human pancreatic lipase corroborate the inhibition mechanism of THL found on the porcine enzyme, and are in full agreement with the identification of the Ser-152 ... His-263 ... Asp-176 catalytic triad in the X-ray structure of human pancreatic lipase.     

Conformational changes induced by ATP-hydrolysis in an ABC transporter: a molecular dynamics study of the Sav1866 exporter

ATP-Binding Cassette (ABC) transporters are ubiquitous membrane proteins that use energy from ATP binding or/and hydrolysis to actively transport allocrites across membranes. In this study, we identify ATP-hydrolysis induced conformational changes in a complete ABC exporter (Sav1866) from Staphylococcus aureaus, using molecular dynamics (MD) simulations. By performing MD simulations on the ATP and ADP+IP bound states, we identify the conformational consequences of hydrolysis, showing that the major rearrangements are not restricted to the NBDs, but extend to the transmembrane domains (TMDs) external regions. For the first time, to our knowledge, we see, within the context of a complete transporter, NBD dimer opening in the ADP+IP state in contrast with all ATP-bound states. This opening results from the dissociation of the ABC signature motif from the nucleotide. In addition, in both states, we observe the opening of a gate entrance in the intracellular loop region leading to the exposure of the TMDs internal cavity to the cytoplasm. To see if this opening was large enough to allow allocrite transport, the adiabatic energy profile for doxorubicin passage was determined. For both states, this profile, although an approximation, is overall downhill from the cytoplasmatic to the extracellular side, and the local energy barriers along the TMDs are relatively small, evidencing the exporter nature of Sav1866. The major difference between states is an energy barrier located in the cytoplasmic gate region, which becomes reduced upon hydrolysis, suggesting that allocrite passage is facilitated, and evidencing a possible molecular mechanism for the active transport in these proteins.     

The mechanism of Klebsiella pneumoniae nitrogenase action. Pre-steady-state kinetics of an enzyme-bound intermediate in N2 reduction and of NH3 formation.

The reduction of N2 to 2NH3 by Klebsiella pneumoniae nitrogenase was studied by a rapid-quench technique. The pre-steady-state time course for N2H4, formed on quenching by the acid-induced hydrolysis of an enzyme-bound intermediate in N2 reduction, showed a 230 ms lag followed by a damped oscillatory approach to a constant concentration in the steady state. The pre-steady-state time course for NH3 formation exhibited a lag of 500 ms and a burst phase that was essentially complete at 1.5s, before a steady-state rate was achieved. These time courses have been simulated by using a previously described kinetic model for the mechanism of nitrogenase action [Lowe & Thorneley (1984) Biochem. J. 224, 877-886]. A hydrazido(2-) structure (=N-NH2) is favoured for the intermediate that yields N2H4 on quenching. The NH3-formation data indicate enzyme-bound metallo-nitrido (identical to N) or -imido (=NH) intermediates formed after N-N bond cleavage to produce the first molecule of NH3 and which subsequently give the second molecule of NH3 by hydrolysis on quenching. The simulations require stoichiometric reduction of one N2 molecule at each Mo and the displacement of one H2 when N2 binds to the MoFe protein. Inhibition by H2 of N2-reduction activity occurs before the formation of the proposed hydrazido(2-) species, and is explained by H2 displacement of N2 at the active site.     

Determination of Ligand Pathways in Globins: APOLAR TUNNELS VERSUS POLAR GATES*

Although molecular dynamics simulations suggest multiple interior pathways for O₂ entry into and exit from globins, most experiments indicate well defined single pathways. In 2001, we highlighted the effects of large-to-small amino acid replacements on rates for ligand entry and exit onto the three-dimensional structure of sperm whale myoglobin. The resultant map argued strongly for ligand movement through a short channel from the heme iron to solvent that is gated by the distal histidine (His-64(E7)) near the solvent edge of the porphyrin ring. In this work, we have applied the same mutagenesis mapping strategy to the neuronal mini-hemoglobin from Cerebratulus lacteus (CerHb), which has a large internal tunnel from the heme iron to the C-terminal ends of the E and H helices, a direction that is 180° opposite to the E7 channel. Detailed comparisons of the new CerHb map with expanded results for Mb show unambiguously that the dominant (>90%) ligand pathway in CerHb is through the internal tunnel, and the major (>75%) ligand pathway in Mb is through the E7 gate. These results demonstrate that: 1) mutagenesis mapping can identify internal pathways when they exist; 2) molecular dynamics simulations need to be refined to address discrepancies with experimental observations; and 3) alternative pathways have evolved in globins to meet specific physiological demands.     

The dynamics of the domains of the IP3-binding site of the inositol-1,4,5-triphosphate-sensitive calcium channel, induced by inositol-1,4,5-triphosphate and calcium

The dynamics of the inositol-1,4,5-triphosphate-sensitive calcium channel after binding of inositol-1,4,5-triphosphate and Ca2+ was analyzed by the Monte Carlo minimization technique. It was shown that the binding of Ca2+ with the unliganded receptor (channel) leads to a turning of the beta-sheet domain relative to the alpha-helical domain with the formation of the receptor conformation that is open for the entry of ions into the cytoplasmic channel vestibule, sterically closed for their passage through the vestibule in the part adjacent to the alpha-helical domains, and unfavourable for subsequent binding of inositol-1,4,5-triphosphate with the receptor. When both co-agonists bind to the receptor, the structure rearrangements induced eliminate both these steric obstacles for the passage of ions through the IP3-binding domain: one at the entrance of the channel cytoplasmic vestibule and the other that is placed deeper in the vestibule near the alpha-domains. The role of the dynamics of the receptor binding core in the IP3-sensitive channel gating is discussed.     

Lipase from Pseudomonas stutzeri: Purification,homology modelling and rational explanation of the substrate binding mode

After purification from the crude commercial preparation, the 3D structure of the synthetically valuable lipase from Pseudomonas stutzeri (LipC) is described through homology modelling, leading to a rational explanation of its catalytic behaviour. This elucidates that the enzyme has an active site defined by residues Ser-109, His-277 and Asp-255, and an oxyanion hole formed by peptidic NH groups from Met-43 and His-110. Interestingly, the active site is covered by two lids, one of them (Lid1, residues 145–181) being larger than the other (Lid2, residues 233–253). The opening and closing of these lids have been simulated by molecular modelling assuming both water and pure THF as solvents. Accordingly, THF clearly helps the exposure of the catalytic serine to the reaction medium which explains its excellent reported performance in this organic solvent. On the other hand, the stereospecificity of this enzyme is explained considering a small hydrophobic cavity formed by Gly-45, Phe-46, Tyr-54, Trp-55, Leu-278, Val-281 and Phe-284; particularly, Tyr-54 plays an important role in substrate recognition. In fact, in benzoin acylation, this residue forces the benzoyl group of the substrate to go into that cavity via H bonding with the carbonyl O atom of benzoin, thereby explaining the observed S-preference in benzoin acylation, which apparently contradicts the canonical Kazlauskas’ rule. For other alcohols non possessing the α-hydroxycarbonyl core, Tyr-54 is allowing the entrance into the above-mentioned hydrophobic cavity only to those substrates with no steric hindrance in the vicinity of the hydroxymethane moiety.     

The apolar channel in Cerebratulus lacteus hemoglobin is the route for O2 entry and exit

The major pathway for O2 binding to mammalian myoglobins (Mb) and hemoglobins (Hb) involves transient upward movement of the distal histidine (His-64(E7)), allowing ligand capture in the distal pocket. The mini-globin from Cerebratulus lacteus (CerHb) appears to have an alternative pathway between the E and H helices that is made accessible by loss of the N-terminal A helix. To test this pathway, we examined the effects of changing the size of the E7 gate and closing the end of the apolar channel in CerHb by site-directed mutagenesis. Increasing the size of Gln-44(E7) from Ala to Trp causes variation of association (k'O2) and dissociation (kO2) rate coefficients, but the changes are not systematic. More significantly, the fractions (Fgem approximately 0.05-0.19) and rates (kgem approximately 50-100 micros(-1)) of geminate CO recombination in the Gln-44(E7) mutants are all similar. In contrast, blocking the entrance to the apolar channel by increasing the size of Ala-55(E18) to Phe and Trp causes the following: 1) both k'O2 and kO2 to decrease roughly 4-fold; 2) Fgem for CO to increase from approximately 0.05 to 0.45; and 3) kgem to decrease from approximately 80 to approximately 9 micros(-1), as ligands become trapped in the channel. Crystal structures and low temperature Fourier-transform infrared spectra of Phe-55 and Trp-55 CerHb confirm that the aromatic side chains block the channel entrance, with little effect on the distal pocket. These results provide unambiguous experimental proof that diatomic ligands can enter and exit a globin through an interior channel in preference to the more direct E7 pathway.     

Shp2 is required for protein kinase C-dependent phosphorylation of serine 307 in insulin receptor substrate-1

The function of insulin receptor substrate-1 (IRS-1), a key molecule of insulin signaling, is modulated by phosphorylation at multiple serine/threonine residues. Phorbol ester stimulation of cells induces phosphorylation of two inhibitory serine residues in IRS-1, i.e. Ser-307 and Ser-318, suggesting that both sites may be targets of protein kinase C (PKC) isoforms. However, in an in vitro system using a broad spectrum of PKC isoforms (alpha, beta1, beta2, delta, epsilon, eta, mu), we detected only Ser-318, but not Ser-307 phosphorylation, suggesting that phorbol ester-induced phosphorylation of this site in intact cells requires additional signaling elements and serine kinases that link PKC activation to Ser-307 phosphorylation. As we have observed recently that the tyrosine phosphatase Shp2, a negative regulator of insulin signaling, is a substrate of PKC, we studied the role of Shp2 in this context. We found that phorbol ester-induced Ser-307 phosphorylation is reduced markedly in Shp2-deficient mouse embryonic fibroblasts (Shp2-/-) whereas Ser-318 phosphorylation is unaltered. The Ser-307 phosphorylation was rescued by transfection of mouse embryonic fibroblasts with wild-type Shp2 or with a phosphatase-inactive Shp2 mutant, respectively. In this cell model, tumor necrosis factor-alpha-induced Ser-307 phosphorylation as well depended on the presence of Shp2. Furthermore, Shp2-dependent phorbol ester effects on Ser-307 were blocked by wortmannin, rapamycin, and the c-Jun NH2-terminal kinase (JNK) inhibitor SP600125. This suggests an involvement of the phosphatidylinositol 3-kinase/mammalian target of rapamycin cascade and of JNK in this signaling pathway resulting in IRS-1 Ser-307 phosphorylation. Because the activation of these kinases does not depend on Shp2, it is concluded that the function of Shp2 is to direct these activated kinases to IRS-1.     

Crystal structure of Escherichia coli penicillin-binding protein 5 bound to a tripeptide boronic acid inhibitor: a role for Ser-110 in deacylation

Penicillin-binding protein 5 (PBP 5) from Escherichia coli is a well-characterized d-alanine carboxypeptidase that serves as a prototypical enzyme to elucidate the structure, function, and catalytic mechanism of PBPs. A comprehensive understanding of the catalytic mechanism underlying d-alanine carboxypeptidation and antibiotic binding has proven elusive. In this study, we report the crystal structure at 1.6 A resolution of PBP 5 in complex with a substrate-like peptide boronic acid, which was designed to resemble the transition-state intermediate during the deacylation step of the enzyme-catalyzed reaction with peptide substrates. In the structure of the complex, the boron atom is covalently attached to Ser-44, which in turn is within hydrogen-bonding distance to Lys-47. This arrangement further supports the assignment of Lys-47 as the general base that activates Ser-44 during acylation. One of the two hydroxyls in the boronyl center (O2) is held by the oxyanion hole comprising the amides of Ser-44 and His-216, while the other hydroxyl (O3), which is analogous to the nucleophilic water for hydrolysis of the acyl-enzyme intermediate, is solvated by a water molecule that bridges to Ser-110. Lys-47 is not well-positioned to act as the catalytic base in the deacylation reaction. Instead, these data suggest a mechanism of catalysis for deacylation that uses a hydrogen-bonding network, involving Lys-213, Ser-110, and a bridging water molecule, to polarize the hydrolytic water molecule.     

cAMP-dependent protein kinase phosphorylation produces interdomain movement in SUR2B leading to activation of the vascular KATP channel

Vascular ATP-sensitive K(+) channels are activated by multiple vasodilating hormones and neurotransmitters via PKA. A critical PKA phosphorylation site (Ser-1387) is found in the second nucleotide-binding domain (NBD(2)) of the SUR2B subunit. To understand how phosphorylation at Ser-1387 leads to changes in channel activity, we modeled the SUR2B using a newly crystallized ABC protein SAV1866. The model showed that Ser-1387 was located on the interface of NBD2 with TMD1 and physically interacted with Tyr-506 in TMD1. A positively charged residue (Arg-1462) in NBD2 was revealed in the close vicinity of Ser-1387. Mutation of either of these three residues abolished PKA-dependent channel activation. Molecular dynamics simulations suggested that Ser-1387, Tyr-506, and Arg-1462 formed a compact triad upon Ser-1387 phosphorylation, leading to reshaping of the NBD2 interface and movements of NBD2 and TMD1. Restriction of the interdomain movements by engineering a disulfide bond between TMD1 and NBD2 prevented the channel activation in a redox-dependent manner. Thus, a channel-gating mechanism is suggested through enhancing the NBD-TMD coupling efficiency following Ser-1387 phosphorylation, which is shared by multiple vasodilators.     

Molecular dynamics simulations and KcsA channel gating

The gating mechanism of a bacterial potassium channel, KcsA, has been investigated via multi-nanosecond molecular dynamic simulations of the channel molecules embedded in a fully solvated palmitoyloleoylphosphatidylcholine bilayer. Four events are seen in which a cation (K(+) or, in one case, Na(+)) initially present in the central cavity exits through the intracellular mouth (the presumed gate) of the channel. Whilst in the cavity a cation interacts with the sidechain T107 O gamma atom of one of the subunits prior to its exit from the channel. Secondary structure analysis as a function of time reveals a break in the helicity of one of the M2 helices. This break is expected to lend flexibility to the helices, enabling them to "open" (minimum pore radius >0.13 nm) and "close" (minimum pore radius <0.13 nm) the channel. Fluctuations in the pore radius at the intracellular gate region are of the order of 0.05 nm, with an average radius in the region of the gate of ca. 0.1 nm. However, around the time of exit of a cation, the pore widens to about 0.15 nm. The distances between the C alpha atoms of the inner helices M2 reveal a coupled increase and decrease between the opposite pair of helices at about the time of exit of the ion. This suggests a breathing motion of the M2 helices that may form the basis for a gating mechanism.     

Structural determinants of lateral gate opening in the protein translocon

The heterotrimeric SecY/Sec61 complex is a protein-conducting channel that provides a passage for proteins across the membrane as well as a means to integrate nascent proteins into the membrane. While the first function is common among membrane protein channels and transporters, the latter is unique. Insertion of nascent membrane proteins, one transmembrane segment at a time, by SecY likely occurs through a lateral gate in the channel. Molecular dynamics simulations have been used to investigate the mechanism of gate opening. Opening and closing the gate under different conditions allowed us to identify structural elements that resist opening as well as those that aid closure. SecE, considered to act as a clamp keeping the lateral gate closed, was found to play no such role. Loosening of the plug by lateral gate opening, a potential step in channel gating, was also observed. The simulations revealed that lipids on time scales of up to 1 micros do not flood channels with an open lateral gate.