Molecular dynamics simulations reveal that apo‐HisJ can sample a closed conformation

The Escherichia coli histidine binding protein HisJ is a type II periplasmic binding protein (PBP) that preferentially binds histidine and interacts with its cytoplasmic membrane ABC transporter, HisQMP₂, to initiate histidine transport. HisJ is a bilobal protein where the larger Domain 1 is connected to the smaller Domain 2 via two linking strands. Type II PBPs are thought to undergo “Venus flytrap” movements where the protein is able to reversibly transition from an open to a closed conformation. To explore the accessibility of the closed conformation to the apo state of the protein, we performed a set of all‐atom molecular dynamics simulations of HisJ starting from four different conformations: apo‐open, apo‐closed, apo‐semiopen, and holo‐closed. The simulations reveal that the closed conformation is less dynamic than the open one. HisJ experienced closing motions and explored semiopen conformations that reverted to closed forms resembling those found in the holo‐closed state. Essential dynamics analysis of the simulations identified domain closing/opening and twisting as main motions. The formation of specific inter‐hinge strand and interdomain polar interactions contributed to the adoption of the closed apo‐conformations although they are up to 2.5‐fold less prevalent compared with the holo‐closed simulations. The overall sampling of the closed form by apo‐HisJ provides a rationale for the binding of unliganded PBPs with their cytoplasmic membrane ABC transporters. Proteins 2014; 82:386–398. © 2013 Wiley Periodicals, Inc.     

A mathematical model on the closing and opening mechanism for Venus flytrap

This paper investigates the opening and closing mechanism for the Venus flytrap (Dionaea muscipula). A mathematical model has been proposed to explain how the flytrap transitions between open, semi-closed and closed states. The model accounts for the charge accumulation of action potentials, which generated by mechanical stimulation of the sensitive trigger hairs on the lobes of the flytrap. Though many studies have been reported for the Venus flytrap opening and closing mechanism, this paper attempts to explain the mechanism from nonlinear dynamics and control perspective.Key words: Venus flytrap, modelling, kinetics     

Study on the mechanism of the BtuF periplasmic-binding protein for vitamin B12

BtuF is the periplasmic binding protein (PBP) that binds vitamin B₁₂ and delivers it to the periplasmic surface of the ABC transporter BtuCD. PBPs generally exhibit considerable conformational changes during ligand binding process, however, BtuF belongs to a subclass of PBPs that, doesn't show such behavior on the basis of the crystal structures. Employing steered molecular dynamics on the B₁₂-bound BtuF, we investigated the energetics and mechanism of BtuF. A potential of mean force along the postulated vitamin B₁₂ unbinding pathway was constructed through Jarzynski's equality. The large free energy differences of the postulated B₁₂ unbinding process suggests the B₁₂-bound structure is in a stable closed state and some conformation changes may be necessary to the B₁₂ unbinding. From the result of the principal component analysis, we found the BtuF-B₁₂ complex shows clear opening-closing and twisting motion tendencies which may facilitate the unbinding of B₁₂ from the binding pocket. The intrinsic flexibility of BtuF was also explored, and it's suggested the Trp44-Gln45 pair, which is situated at the mouth of the B₁₂ binding pocket, may act as a gate in the B₁₂ binding and unbinding process.     

Trapping open and closed forms of FitE—A group III periplasmic binding protein

Periplasmic binding proteins (PBPs) are essential components of bacterial transport systems, necessary for bacterial growth and survival. The two‐domain structures of PBPs are topologically classified into three groups based on the number of crossovers or hinges between the globular domains: group I PBPs have three connections, group II have two, and group III have only one. Although a large number of structures for group I or II PBPs are known, fewer group III PBPs have been structurally characterized. Group I and II PBPs exhibit significant domain motions during transition from the unbound to ligand‐bound form, however, no large conformational changes have been observed to date in group III PBPs. We have solved the crystal structure of a periplasmic binding protein FitE, part of an iron transport system, fit, recently identified in a clinical E. coli isolate. The structure, determined at 1.8 Å resolution, shows that FitE is a group III PBP containing a single α‐helix bridging the two domains. Among the individual FitE molecules present in two crystal forms we observed three different conformations (open, closed, intermediate). Our crystallographic and molecular dynamics results strongly support the notion that group III PBPs also adopt the same Venus flytrap mechanism as do groups I and II PBPs. Unlike other group III PBPs, FitE forms dimers both in solution and in the crystals. The putative siderophore binding pocket is lined with arginine residues, suggesting an anionic nature of the iron‐containing siderophore. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Crystal structures of the BtuF periplasmic-binding protein for vitamin B12 suggest a functionally important reduction in protein mobility upon ligand binding

BtuF is the periplasmic binding protein (PBP) for the vitamin B12 transporter BtuCD, a member of the ATP-binding cassette (ABC) transporter superfamily of transmembrane pumps. We have determined crystal structures of Escherichia coli BtuF in the apo state at 3.0 A resolution and with vitamin B12 bound at 2.0 A resolution. The structure of BtuF is similar to that of the FhuD and TroA PBPs and is composed of two alpha/beta domains linked by a rigid alpha-helix. B12 is bound in the "base-on" or vitamin conformation in a wide acidic cleft located between these domains. The C-terminal domain shares structural homology to a B12-binding domain found in a variety of enzymes. The same surface of this domain interacts with opposite surfaces of B12 when comparing ligand-bound structures of BtuF and the homologous enzymes, a change that is probably caused by the obstruction of the face that typically interacts with this domain by the base-on conformation of vitamin B12 bound to BtuF. There is no apparent pseudo-symmetry in the surface properties of the BtuF domains flanking its B12 binding site even though the presumed transport site in the previously reported crystal structure of BtuCD is located in an intersubunit interface with 2-fold symmetry. Unwinding of an alpha-helix in the C-terminal domain of BtuF appears to be part of conformational change involving a general increase in the mobility of this domain in the apo structure compared with the B12-bound structure. As this helix is located on the surface likely to interact with BtuC, unwinding of the helix upon binding to BtuC could play a role in triggering release of B12 into the transport cavity. Furthermore, the high mobility of this domain in free BtuF could provide an entropic driving force for the subsequent release of BtuF required to complete the transport cycle.     

Holo‐BtuF stabilizes the open conformation of the vitamin B12 ABC transporter BtuCD

While there is evidence that other ABC transporters can tell between empty and loaded substrate binding protein, reconstitution experiments suggest otherwise for the Escherichia coli vitamin B12 importer BtuCD‐F. Here, we address the question of BtuCD‐F substrate sensitivity in a combined protein–protein docking and molecular dynamics simulation approach. Starting from the BtuCD and holo‐BtuF crystal structures, we model two holo‐BtuCD‐F docking complexes differing by a 180° orientation of BtuF. One of these is similar to the apo‐BtuCD‐F crystal structure. Both docking complexes were embedded in a lipid/water environment to investigate their dynamics and BtuCD's conformational response to the presence and absence of BtuF, vitamin B12, and Mg‐ATP in a series of 28 independent MD simulations. We find holo‐BtuF stabilizing the open conformation of BtuCD, whereas the transporter begins to close again when BtuF or vitamin B12 is removed—suggesting BtuCD‐F is capable of substrate sensitivity. We identified BtuC transmembrane helices 3 and 5, the L‐loops and the adjacent helices comprised of BtuC residues 170–180 as hotspots of conformational change. We propose the latter to act as substrate sensors. BtuF‐Trp44 appears to act as a lid on the vitamin B12 binding cleft in BtuF X‐ray structures and protrudes into the BtuCD transport channel in one of our simulations, which might represent an initial step in vitamin B12 uptake. On an average, we observe subunit motions where the nucleotide binding domains approach each other while the transmembrane domains display an opening trend toward the periplasm. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Molecular dynamics simulations of the conformational changes of the glutamate receptor ligand-binding core in the presence of glutamate and kainate

Excitatory synaptic transmission is mediated by ionotropic glutamate receptors (iGluRs) through the induced transient opening of transmembrane ion channels. The three-dimensional structure of the extracellular ligand-binding core of iGluRs shares the overall features of bacterial periplasmic binding proteins (PBPs). In both families of proteins, the ligand-binding site is arranged in two domains separated by a cleft and connected by two peptide stretches. PBPs undergo a typical hinge motion of the two domains associated with ligand binding that leads to a conformational change from an open to a closed form. The common architecture suggests a similar closing mechanism in the ligand-binding core of iGluRs induced by the binding of specific agonists. Starting from the experimentally determined kainate-bound closed form of the S1S2 GluR2 construct, we have studied by means of molecular dynamics simulations the opening motion of the ligand-binding core in the presence and in the absence of both glutamate and kainate. Our results suggest that the opening/closing interdomain hinge motions are coupled to conformational changes in the insertion region of the transmembrane segments. These changes are triggered by the interaction of the agonists with the essential Glu 209 residue. A plausible mechanism for the coupling of agonist binding to channel gating is discussed.     

The Solution Structure,Binding Properties,and Dynamics of the Bacterial Siderophore-binding Protein FepB

The periplasmic binding protein (PBP) FepB plays a key role in transporting the catecholate siderophore ferric enterobactin from the outer to the inner membrane in Gram-negative bacteria. The solution structures of the 34-kDa apo- and holo-FepB from Escherichia coli, solved by NMR, represent the first solution structures determined for the type III class of PBPs. Unlike type I and II PBPs, which undergo large “Venus flytrap” conformational changes upon ligand binding, both forms of FepB maintain similar overall folds; however, binding of the ligand is accompanied by significant loop movements. Reverse methyl cross-saturation experiments corroborated chemical shift perturbation results and uniquely defined the binding pocket for gallium enterobactin (GaEnt). NMR relaxation experiments indicated that a flexible loop (residues 225–250) adopted a more rigid and extended conformation upon ligand binding, which positioned residues for optimal interactions with the ligand and the cytoplasmic membrane ABC transporter (FepCD), respectively. In conclusion, this work highlights the pivotal role that structural dynamics plays in ligand binding and transporter interactions in type III PBPs.     

Closing of Venus Flytrap by Electrical Stimulation of Motor Cells

Electrical signaling and rapid closure of the carnivorous plant Dionaea muscipula Ellis (Venus flytrap) have been attracting the attention of researchers since XIX century, but the exact mechanism of Venus flytrap closure is still unknown. We found that the electrical stimulus between a midrib and a lobe closes the Venus flytrap leaf by activating motor cells without mechanical stimulation of trigger hairs. The closing time of Venus flytrap by electrical stimulation of motor cells is 0.3 s, the same as mechanically induced closing. The mean electrical charge required for the closure of the Venus flytrap leaf is 13.6 µC. Ion channel blockers such as Ba²⁺, TEACl as well as uncouplers such as FCCP, 2,4-dinitrophenol and pentachlorophenol dramatically decrease the speed of the trap closing. Using an ultra-fast data acquisition system with measurements in real time, we found that the action potential in the Venus flytrap has a duration time of about 1.5 ms. Our results demonstrate that electrical stimulation can be used to study mechanisms of fast activity in motor cells of the plant kingdom.Key Words: action potential, electrophysiology, electrical signaling, Venus flytrap, motor cells     

Amino acid recognition by Venus flytrap domains is encoded in an 8-residue motif

A motif foramino acid recognition by proteins or domains of the periplasmic binding protein-like I superfamily has been identified. An initial pattern of 5 residues was based on a multiple sequence alignment of selected proteins of that fold family and on common structural features observed in the crystal structure of some members of the family [leucine isoleucine valine binding protein (LIVBP), leucine binding protein (LBP), and metabotropic glutamate receptor type 1 (mGlu1R) amino terminal domain)]. This pattern was used against the PIR-NREF sequence database and further refined to retrieve all sequences of proteins that belong to the family and eliminate those that do not belong to it. A motif of 8 residues was finally selected to build up the general signature. A total of 232 sequences were retrieved. They were found to belong to only three families of proteins: bacterial periplasmic binding proteins (PBP, 71 sequences), family 3 (or C) of G-protein coupled receptor (GPCR) (146 sequences), and plant putative ionotropic glutamate receptors (iGluR, 15 sequences). PBPs are known to adopt a bilobate structure also named Venus flytrap domain, or LIVBP domain in the present case. Family 3/C GPCRs are also known to hold such a domain. However, for plant iGluRs, it was previously detected by classical similarity searches but not specifically described. Thus plant iGluRs carry two Venus flytrap domains, one that binds glutamate and an additional one that would be a modulatory LIVBP domain. In some cases, the modulator binding to that domain would be an amino acid.     

Molecular dynamics simulations of the bacterial periplasmic heme binding proteins ShuT and PhuT

ShuT and PhuT are two periplasmic heme binding proteins that shuttle heme between the outer and inner membranes of the Gram-negative bacteria. Periplasmic binding proteins (PBPs) generally exhibit considerable conformational changes during the ligand binding process, whereas ShuT and PhuT belong to a class of PBPs that do not show such behavior based on their apo and holo crystal structures. By employing a series of molecular dynamic simulations on the ShuT and the PhuT, the dynamics and functions of the two PBPs were investigated. Through monitoring the distance changes between the two conserved glutamates of ShuT and PhuT, it was found the two PBPs were more flexible than previously assumed, exhibiting obvious opening-closing motions which were more remarkable in the apo runs of ShuT. Based on the results of the domain motion analysis, large scale conformational transitions were found in all apo runs of ShuT and PhuT, hinting that the domain motions of the two PBPs may be intrinsic. On the basis of the results of the principle component analysis, distinct opening-closing and twisting motion tendencies were observed not only in the apo, but also in the holo simulations of the two PBPs. The Gaussian network model was applied in order to analyze the hinge bending regions. The most important bending regions of ShuT and PhuT are located around the midpoints of their respective connecting helixes. Finally, the flexibilities and the details of the simulations of ShuT and PhuT were discussed. Characterized by the remarkably large flexibilities, the loop constituted by Ala 169, Gly170 and Gly171 of ShuT and the beta-turn constituted by Ala176, Gly177 and Gly178 of PhuT may be important for the functions of the two PBPs. Furthermore, the Asn254 of ShuT and the Arg228 of PhuT may be indispensable for the binding or unbinding of heme, since it is involved in the important hydrogen bonding to the propionate side-chains of heme.     

Complete hunting cycle of Dionaea muscipula: Consecutive steps and their electrical properties

The total hunting cycle of the Venus flytrap consists of five stages: 1. Open state → 2. Closed state → 3. Locked state → 4. Constriction and digestion → 5. Semi-open state → 1. Open state. The opening of the trap after digestion consists of two steps: opening of the lobes, and changing of their curvature from concave to convex shape. Uncouplers carbonylcyanide-4-trifluoromethoxyphenyl hydrazone (FCCP) and carbonylcyanide-3-chlorophenylhydrazone (CCCP) inhibit the trap from opening for two weeks and antracene-9-carboxylic acid inhibits the trap from constricting. Different stages of the hunting cycle have different electrical characteristics. The biologically closed electrochemical circuits in the Venus flytrap are analyzed using the charged capacitor method. If the initial voltage applied to the Venus flytrap is 0.5 V or greater, changing the polarity of the electrodes between the midrib and one of the lobes results in a rectification effect and in different kinetics of discharge capacitance. These effects can be caused by the fast transport of ions through ion channels. The electrical properties of the Venus flytrap were investigated and equivalent electrical circuits within the upper leaf were proposed to explain the experimental data.     

Molecular mechanism of the allosteric enhancement of the umami taste sensation

The fifth taste quality, umami, arises from binding of glutamate to the umami receptor T1R1/T1R3. The umami taste is enhanced several-fold upon addition of free nucleotides such as guanosine-5'-monophosphate (GMP) to glutamate-containing food. GMP may operate via binding to the ligand-binding domain of the T1R1 part of the umami receptor at an allosteric site. Using molecular dynamics simulations, we show that GMP can stabilize the closed (active) state of T1R1 by binding to the outer vestibule of the so-called Venus flytrap domain of the receptor. The transition between the closed and open conformations was accessed in the simulations. Using principal component analysis, we show that the dynamics of the Venus flytrap domain along the hinge-bending motion that activates signaling is dampened significantly upon binding of glutamate, and further slows down upon binding of GMP at an allosteric site, thus suggesting a molecular mechanism of cooperativity between GMP and glutamate.     

Substrate Binding Specifically Modulates Domain Arrangements in Adenylate Kinase

The enzyme adenylate kinase (ADK) features two substrate binding domains that undergo large-scale motions during catalysis. In the apo state, the enzyme preferentially adopts a globally open state with accessible binding sites. Binding of two substrate molecules (AMP + ATP or ADP + ADP) results in a closed domain conformation, allowing efficient phosphoryl-transfer catalysis. We employed molecular dynamics simulations to systematically investigate how the individual domain motions are modulated by the binding of substrates. Two-dimensional free-energy landscapes were calculated along the opening of the two flexible lid domains for apo and holo ADK as well as for all single natural substrates bound to one of the two binding sites of ADK. The simulations reveal a strong dependence of the conformational ensembles on type and binding position of the bound substrates and a nonsymmetric behavior of the lid domains. Altogether, the ensembles suggest that, upon initial substrate binding to the corresponding lid site, the opposing lid is maintained open and accessible for subsequent substrate binding. In contrast, ATP binding to the AMP-lid induces global domain closing, preventing further substrate binding to the ATP-lid site. This might constitute a mechanism by which the enzyme avoids the formation of a stable but enzymatically unproductive state.     

Crystal Structures of Penicillin-Binding Protein 3 (PBP3) from Methicillin-Resistant Staphylococcus aureus in the Apo and Cefotaxime‐Bound Forms

Staphylococcus aureus is a widespread Gram‐positive opportunistic pathogen, and a methicillin‐resistant form (MRSA) is particularly difficult to treat clinically. We have solved two crystal structures of penicillin‐binding protein (PBP) 3 (PBP3) from MRSA, the apo form and a complex with the β-lactam antibiotic cefotaxime, and used electrospray mass spectrometry to measure its sensitivity to a variety of penicillin derivatives. PBP3 is a class B PBP, possessing an N-terminal non-penicillin‐binding domain, sometimes called a dimerization domain, and a C-terminal transpeptidase domain. The model shows a different orientation of its two domains compared to earlier models of other class B PBPs and a novel, larger N-domain. Consistent with the nomenclature of “dimerization domain”, the N-terminal region forms an apparently tight interaction with a neighboring molecule related by a 2-fold symmetry axis in the crystal structure. This dimer form is predicted to be highly stable in solution by the PISA server, but mass spectrometry and analytical ultracentrifugation provide unequivocal evidence that the protein is a monomer in solution.     

Crystal structures of penicillin-binding protein 3 from Pseudomonas aeruginosa: comparison of native and antibiotic-bound forms

We report the first crystal structures of a penicillin-binding protein (PBP), PBP3, from Pseudomonas aeruginosa in native form and covalently linked to two important β-lactam antibiotics, carbenicillin and ceftazidime. Overall, the structures of apo and acyl complexes are very similar; however, variations in the orientation of the amino-terminal membrane-proximal domain relative to that of the carboxy-terminal transpeptidase domain indicate interdomain flexibility. Binding of either carbenicillin or ceftazidime to purified PBP3 increases the thermostability of the enzyme significantly and is associated with local conformational changes, which lead to a narrowing of the substrate-binding cleft. The orientations of the two β-lactams in the active site and the key interactions formed between the ligands and PBP3 are similar despite differences in the two drugs, indicating a degree of flexibility in the binding site. The conserved binding mode of β-lactam-based inhibitors appears to extend to other PBPs, as suggested by a comparison of the PBP3/ceftazidime complex and the Escherichia coli PBP1b/ceftoxamine complex. Since P. aeruginosa is an important human pathogen, the structural data reveal the mode of action of the frontline antibiotic ceftazidime at the molecular level. Improved drugs to combat infections by P. aeruginosa and related Gram-negative bacteria are sought and our study provides templates to assist that process and allows us to discuss new ways of inhibiting PBPs.     

Crystal Structures of Penicillin-Binding Proteins 4 and 5 from Haemophilus influenzae

We have determined high-resolution apo crystal structures of two low molecular weight penicillin-binding proteins (PBPs), PBP4 and PBP5, from Haemophilus influenzae, one of the most frequently found pathogens in the upper respiratory tract of children. Novel β-lactams with notable antimicrobial activity have been designed, and crystal structures of PBP4 complexed with ampicillin and two of the novel molecules have also been determined. Comparing the apo form with those of the complexes, we find that the drugs disturb the PBP4 structure and weaken X-ray diffraction, to very different extents. PBP4 has recently been shown to act as a sensor of the presence of penicillins in Pseudomonas aeruginosa, and our models offer a clue to the structural basis for this effect. Covalently attached penicillins press against a phenylalanine residue near the active site and disturb the deacylation step. The ready inhibition of PBP4 by β-lactams compared to PBP5 also appears to be related to the weaker interactions holding key residues in a catalytically competent position.     

A PBP2x from a clinical isolate of Streptococcus pneumoniae exhibits an alternative mechanism for reduction of susceptibility to beta-lactam antibiotics

The human pathogen Streptococcus pneumoniae is one of the main causative agents of respiratory tract infections. At present, clinical isolates of S. pneumoniae often exhibit decreased susceptibility toward beta-lactams, a phenomenon linked to multiple mutations within the penicillin-binding proteins (PBPs). PBP2x, one of the six PBPs of S. pneumoniae, is the first target to be modified under antibiotic pressure. By comparing 89 S. pneumoniae PBP2x sequences from clinical and public data bases, we have identified one major group of sequences from drug-sensitive strains as well as two distinct groups from drug-resistant strains. The first group includes proteins that display high similarity to PBP2x from the well characterized resistant strain Sp328. The second group includes sequences in which a signature mutation, Q552E, is found adjacent to the third catalytic motif. In this work, a PBP2x from a representative strain from the latter group (S. pneumoniae 5259) was biochemically and structurally characterized. Phenotypical analyses of transformed pneumococci show that the Q552E substitution is responsible for most of the reduction of strain susceptibility toward beta-lactams. The crystal structure of 5259-PBP2x reveals a change in polarity and charge distribution around the active site cavity, as well as rearrangement of strand beta3, emulating structural changes observed for other PBPs that confer drug resistance to Gram-positive pathogens. Interestingly, the active site of 5259-PBP2x is in closed conformation, whereas that of Sp328-PBP2x is open. Consequently, S. pneumoniae has evolved to employ the same protein in two distinct mechanisms of antibiotic resistance.