Macrolide-specific efflux pump MacAB-TolC has been identified in diverse Gram-negative bacteria including Escherichia coli. The inner membrane transporter MacB requires the outer membrane factor TolC and the periplasmic adaptor protein MacA to form a functional tripartite complex. In this study, we used a chimeric protein containing the tip region of the TolC ␣-barrel to investigate the role of the TolC ␣-barrel tip region with regard to its interaction with MacA. The chimeric protein formed a stable complex with MacA, and the complex formation was abolished by substitution at the functionally essential residues located at the MacA ␣-helical tip region. Electron microscopic study delineated that this complex was made by tip-to-tip interaction between the tip regions of the ␣-barrels of TolC and MacA, which correlated well with the TolC and MacA complex calculated by molecular dynamics. Taken together, our results demonstrate that the MacA hexamer interacts with TolC in a tip-to-tip manner, and implies the manner by which MacA induces opening of the TolC channel.Drug resistance of microbial pathogens presents an increasing threat to public health (1). In Gram-negative pathogens, high levels of intrinsic or acquired drug resistance are conferred by three-component multidrug efflux pumps, which are composed of the inner membrane transporter, the outer membrane factor (OMF), and the periplasmic membrane fusion protein (MFP) 4 (2-5). These tripartite complexes span the entire twomembrane envelope of Gram-negative bacteria and expel various molecules into the medium, utilizing a proton gradient or ATP hydrolysis. The inner membrane transporters belong to one of three structurally dissimilar superfamilies of proteins: resistance-nodulation-cell division (RND), ATP-binding cassette (ABC), or major facilitator. The inner membrane transporters expel the substrates through the central channel of the OMF, as exemplified by Escherichia coli TolC, which spans the outer membrane (6). The MFP, which connects the other two components in the periplasm, is also essential for the function of the efflux pump.In E. coli, AcrAB-TolC acts as a major multidrug efflux pump (7-9), where AcrB is the RND-type inner membrane transporter and AcrA belongs to MFP. The homotrimeric TolC is embedded in the outer membrane and continues ϳ100 Å into the periplasmic space as an ␣-barrel composed of six ␣-hairpins that form the wall of a 35-Å inner-diameter cylindrical channel (10). The TolC channel is closed at the aperture end and the channel opening is induced only in the presence of the other components, the mechanism of which remains to be determined at the molecular level.The MacAB-TolC pump has been identified in E. coli; the inner membrane transporter MacB belongs to non-canonic ABC-type transporters (8,9,11,12), and MFP MacA shares structural similarity with AcrA (sequence similarity 44%) (13). Overproduction of MacAB results in increased resistance to the macrolide antibiotics in macrolide-susceptible AcrAB-deficient E. coli (8, 9, 11).The s...
Long-time molecular dynamics (MD) simulations are now able to fold small proteins reversibly to their native structures [LindorffLarsen K, Piana S, Dror RO, Shaw DE (2011) Science 334(6055):517-520]. These results indicate that modern force fields can reproduce the energy surface near the native structure. To test how well the force fields recapitulate the other regions of the energy surface, MD trajectories for a variant of protein G are compared with data from site-resolved hydrogen exchange (HX) and other biophysical measurements. Because HX monitors the breaking of individual H-bonds, this experimental technique identifies the stability and H-bond content of excited states, thus enabling quantitative comparison with the simulations. Contrary to experimental findings of a cooperative, all-or-none unfolding process, the simulated denatured state ensemble, on average, is highly collapsed with some transient or persistent native 2°structure. The MD trajectories of this protein G variant and other small proteins exhibit excessive intramolecular H-bonding even for the most expanded conformations, suggesting that the force fields require improvements in describing H-bonding and backbone hydration. Moreover, these comparisons provide a general protocol for validating the ability of simulations to accurately capture rare structural fluctuations. M olecular dynamics (MD) simulations can now probe protein dynamics on millisecond timescales and thereby enable investigation of a variety of biological problems, including binding, conformational changes, and folding. A landmark example is the all-atom simulations by Shaw and coworkers where multiple folding and unfolding events were observed in long time trajectories (1, 2). In addition to predicting or matching observed folding rates with a single set of parameters, these simulations produced native-like models for 12 small, fast-folding proteins. Equally impressive is their observation of multiple discrete folding and unfolding transitions, which indicates that folding proceeds on an energy landscape with two major states separated by a free energy barrier. This barrier-limited folding behavior replicates that observed for many proteins. Not surprisingly, these remarkable simulations are being extensively analyzed (3-5).The applicability of MD for many situations is limited by the extent to which the entire landscape is recapitulated. An accurate representation of native-like states does not imply a correct representation of other states (e.g., intermediates and unfolded structures). Proper validation requires a comparison with experiments that probe lowly populated conformations. NMR measurements probe subsecond dynamics with single residue resolution, although with a limitation to states with populations exceeding 0.5% (6). Fluorescence, CD, FRET, and small angle X-ray scattering (SAXS) measurements are well adapted to kinetic studies but provide limited spatial resolution.Hydrogen exchange (HX) data report on the H-bond patterns and populations of extremely rare state...
The burial of hydrophobic side chains in a protein core generally is thought to be the major ingredient for stable, cooperative folding. Here, we show that, for the snow flea antifreeze protein (sfAFP), stability and cooperativity can occur without a hydrophobic core, and without α-helices or β-sheets. sfAFP has low sequence complexity with 46% glycine and an interior filled only with backbone H-bonds between six polyproline 2 (PP2) helices. However, the protein folds in a kinetically two-state manner and is moderately stable at room temperature. We believe that a major part of the stability arises from the unusual match between residue-level PP2 dihedral angle bias in the unfolded state and PP2 helical structure in the native state. Additional stabilizing factors that compensate for the dearth of hydrophobic burial include shorter and stronger H-bonds, and increased entropy in the folded state. These results extend our understanding of the origins of cooperativity and stability in protein folding, including the balance between solvent and polypeptide chain entropies.protein folding | cooperativity | kinetics | PP2 | hydrogen bonding T he basis of protein-folding stability and cooperativity remains a topic of great interest (1, 2). Most studies have focused on proteins with hydrophobic cores containing α-helices and β-sheets. Here, we study the folding of snow flea antifreeze protein (sfAFP), a globular protein that lacks these features. The 81-residue protein has a novel fold that is distinct from other proteins (3-6), containing only polyproline 2 (PP2) helices and turns with a core filled with H-bonds and no hydrophobic groups (Fig. 1).The H-bond network between the PP2 helices (6), PP2 1-6 , requires close packing, which would be precluded by the presence of a C β atom. Thus, the PP2 helices are defined by glycine (Gly) repeats (GXX or GGX, where X is any other amino acid), which enable the hydrogen-bond network. As a consequence, the sfAFP molecule has a low-complexity glycine-rich sequence [37/81 Gly residues, or 46%; typical is 6, or 7% (7)]. The sfAFP core is well packed with essentially no interior void volumes, even when probed using a 0.5-Å radius sphere (8). sfAFP also contains two disulfide bonds, C1-C28 and C13-C43.Notably, no side chains are buried in sfAFP's core (2). All 12 hydrophobic side chains (V 4 , K 2 , and P 6 ) are surface exposed. Upon folding, sfAFP buries about 20 Å 2 ·res −1 of hydrophobic surface area compared with an average of 50 Å 2 ·res −1 for a set of 34 proteins of similar size (Fig. 2A). The difference equates to a decrease in stability of ∼1 or 1.4 kcal·mol −1 ·res −1 , assuming a surface tension coefficient of γ ∼ 34 or 47 cal·mol −1 ·Å −2 based on classical (9) or Flory-Huggins theory (10), respectively. Even the lower bound of 1 kcal·mol −1 ·res −1 represents a significant stability loss, and it is not obvious which energetic terms could compensate for the reduction in hydrophobic burial.Because hydrophobic burial is generally regarded as the basis of protein stability a...
PACSY (Protein structure And Chemical Shift NMR spectroscopY) is a relational database management system that integrates information from the Protein Data Bank, the Biological Magnetic Resonance Data Bank, and the Structural Classification of Proteins database. PACSY provides three-dimensional coordinates and chemical shifts of atoms along with derived information such as torsion angles, solvent accessible surface areas, and hydrophobicity scales. PACSY consists of six relational table types linked to one another for coherence by key identification numbers. Database queries are enabled by advanced search functions supported by an RDBMS server such as MySQL or PostgreSQL. PACSY enables users to search for combinations of information from different database sources in support of their research. Two software packages, PACSY Maker for database creation and PACSY Analyzer for database analysis, are available from http://pacsy.nmrfam.wisc.edu.
Experimental and computational folding studies of Proteins L & G and NuG2 typically find that sequence differences determine which of the two hairpins is formed in the transition state ensemble (TSE). However, our recent work on Protein L finds that its TSE contains both hairpins, compelling a reassessment of the influence of sequence on the folding behavior of the other two homologs. We characterize the TSEs for Protein G and NuG2b, a triple mutant of NuG2, using ψ analysis, a method for identifying contacts in the TSE. All three homologs are found to share a common and near-native TSE topology with interactions between all four strands. However, the helical content varies in the TSE, being largely absent in Proteins G & L but partially present in NuG2b. The variability likely arises from competing propensities for the formation of nonnative β turns in the naturally occurring proteins, as observed in our TerItFix folding algorithm. All-atom folding simulations of NuG2b recapitulate the observed TSEs with four strands for 5 of 27 transition paths [Lindorff-Larsen K, Piana S, Dror RO, Shaw DE (2011) Science 334(6055):517–520]. Our data support the view that homologous proteins have similar folding mechanisms, even when nonnative interactions are present in the transition state. These findings emphasize the ongoing challenge of accurately characterizing and predicting TSEs, even for relatively simple proteins.
Cannabinoid receptor 1 (CB1) is a promising therapeutic target for a variety of disorders. Distinct efficacy profiles showed different therapeutic effects on CB1 dependent on three classes of ligands: agonists, antagonists, and inverse agonists. To discriminate the distinct efficacy profiles of the ligands, we carried out molecular dynamics (MD) simulations to identify the dynamic behaviors of inactive and active conformations of CB1 structures with the ligands. In addition, the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method was applied to analyze the binding free energy decompositions of the CB1-ligand complexes. With these two methods, we found the possibility that the three classes of ligands can be discriminated. Our findings shed light on the understanding of different efficacy profiles of ligands by analyzing the structural behaviors of intact CB1 structures and the binding energies of ligands, thereby yielding insights that are useful for the design of new potent CB1 drugs.
Recent experiments claiming that Naf-BBL protein follows a global downhill folding raised an important controversy as to the folding mechanism of fast-folding proteins. Under the global downhill folding scenario, not only do proteins undergo a gradual folding, but folding events along the continuous folding pathway also could be mapped out from the equilibrium denaturation experiment. Based on the exact calculation using a free energy landscape, relaxation eigenmodes from a master equation, and Monte Carlo simulation of an extended Muñoz-Eaton model that incorporates multiscale-heterogeneous pairwise interactions between amino acids, here we show that the very nature of a two-state cooperative transition such as a bimodal distribution from an exact free energy landscape and biphasic relaxation kinetics manifest in the thermodynamics and folding-unfolding kinetics of BBL and peripheral subunit-binding domain homologues. Our results provide an unequivocal resolution to the fundamental controversy related to the global downhill folding scheme, whose applicability to other proteins should be critically reexamined.protein folding mechanism ͉ global downhill folding ͉ protein thermodynamics ͉ relaxation kinetics of protein O ur understanding of protein folding is primarily based on interpretation of the thermodynamics and kinetics from a simple protein folding model (1-10). The principle of minimal frustration and structural consistency captured by the Go model allowed us to describe the complicated nature of protein folding at a coarse-grained level (5, 6). Bryngelson et al. (6,7) posited a phenomenological picture of free energy landscape of a protein-like heteropolymer and classified protein folding into three scenarios depending on whether the free energy landscape is unistable or bistable and also whether a glass transition occurs. Shakhnovich and Gutin (8) presented microscopic statisticalmechanical theories for heteropolymer model, whose consequences provided important insights into the thermodynamics, kinetics, and nature of free energy landscape for protein folding. The conventional view of protein folding assumes a discrete two-state transition between a native state and a denatured ensemble of protein conformations that are separated by a folding barrier (9). The other view termed as one-state (or global) downhill folding asserts that there is a continuous change of protein conformation during folding without distinct free energy barrier (7, 10). The former could be understood as the first order (cooperative) phase transition whereas the latter as the second order (noncooperative) phase transition between a native and a denatured ensemble of protein conformation. An in-depth analysis for possible downhill folding was performed by Abkevich et al. (10) where it was shown that the major determinant for the cooperativity of protein folding is the relative abundance of nonlocal versus local contacts between residues in the native structure of a protein, which also simultaneously compete with the loss of conform...
The cannabinoid receptor 1 (CB1) is a class A G-protein coupled receptor (GPCR) that can exert various effects on the human body through the endocannabinoid system. Understanding CB1 activation has many benefits for the medical use of cannabinoids. A previous study reported that CB1 has two notable residues referred to as the toggle switch, F3.36 and W6.48, which are important for its activation mechanism. We performed a molecular dynamics simulation with a mutation in the toggle switch to examine its role in active and inactive states. We also examined structural changes, the residue–residue interaction network, and the interaction network among helices and loops of wildtype and mutant CB1 for both activation states. As a result, we found that the energetic changes in the hydrogen-bond network of the Na+ pocket, extracellular N-terminus–TM2–ECL1–TM3 interface including D2.63–K3.28 salt-bridge, and extracellular ECL2–TM5–ECL3–TM6 interface directly linked to the toggle switch contribute to the stability of CB1 by the broken aromatic interaction of the toggle switch. It makes the conformation of inactive CB1 receptor to be unstable. Our study explained the role of the toggle switch regarding the energetic interactions related to the Na+ pocket and extracellular loop interfaces, which could contribute to a better understanding of the activation mechanism of CB1.
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