Coarse-Grained Force Fields from the Perspective of Statistical Mechanics: Better Understanding of the Origins of a MARTINI Hangover.

The popular MARTINI coarse-grained model is used as a test case to analyze the adherence of top-down coarse-grained molecular dynamics models (i.e., models primarily parametrized to match experimental results) to the known features of statistical mechanics for the underlying all-atom representations. Specifically, the temperature dependence of various pair distribution functions, and hence their underlying potentials of mean force via the reversible work theorem, are compared between MARTINI 2.0, Dry MARTINI, and all-atom simulations mapped onto equivalent coarse-grained sites for certain lipid bilayers. It is found that the MARTINI models do not completely capture the lipid structure seen in atomistic simulations as projected onto the coarse-grained mappings and that issues of accuracy and temperature transferability arise due to an incorrect enthalpy-entropy decomposition of these potentials of mean force. The potential of mean force for the association of two amphipathic helices in a lipid bilayer is also calculated, and especially at shorter ranges, the MARTINI and all-atom projection results differ substantially. The former is much less repulsive and hence will lead to a higher probability of MARTINI helix association in the MARTINI bilayer than occurs in the actual all-atom case. Additionally, the bilayer height fluctuation spectra are calculated for the MARTINI model, and compared to the all-atom results, it is found that the magnitude of thermally averaged amplitudes at intermediate length scales are quite different, pointing to a number of possible consequences for realistic modeling of membrane processes. Taken as a whole, the results presented here show disagreement in the enthalpic and entropic driving forces driving lateral structure in lipid bilayers as well as quantitative differences in association of embedded amphipathic helices, which can help direct future efforts to parametrize CG models with better agreement to the all-atom systems they aspire to represent.

The importance of conserved amino acids in heme-based globin-coupled diguanylate cyclases.

Globin-coupled diguanylate cyclases contain globin, middle, and diguanylate cyclase domains that sense O2 to synthesize c-di-GMP and regulate bacterial motility, biofilm formation, and virulence. However, relatively few studies have extensively examined the roles of individual residues and domains of globin-coupled diguanylate cyclases, which can shed light on their signaling mechanisms and provide drug targets. Here, we report the critical residues of two globin-coupled diguanylate cyclases, EcGReg from Escherichia coli and BpeGReg from Bordetella pertussis, and show that their diguanylate cyclase activity requires an intact globin domain. In the distal heme pocket of the globin domain, residues Phe42, Tyr43, Ala68 (EcGReg)/Ser68 (BpeGReg), and Met69 are required to maintain full diguanylate cyclase activity. The highly conserved amino acids His223/His225 and Lys224/Lys226 in the middle domain of EcGReg/BpeGReg are essential to diguanylate cyclase activity. We also identified sixteen important residues (Leu300, Arg306, Asp333, Phe337, Lys338, Asn341, Asp342, Asp350, Leu353, Asp368, Arg372, Gly374, Gly375, Asp376, Glu377, and Phe378) in the active site and inhibitory site of the diguanylate cyclase domain of EcGReg. Moreover, BpeGReg266 (residues 1-266) and BpeGReg296 (residues 1-296), which only contain the globin and middle domains, can inhibit bacterial motility. Our findings suggest that the distal residues of the globin domain affect diguanylate cyclase activity and that BpeGReg may interact with other c-di-GMP-metabolizing proteins to form mixed signaling teams.

Molecular dynamics study of hell's gate globin I (HGbI) from a methanotrophic extremophile: oxygen migration through a large cavity.

Hell's gate globin I (HGbI), a heme-containing protein from the extremophile Methylacidiphilum infernorum, has fast oxygen-binding/slow release characteristics due to its distal residues Gln and Tyr. The combination of Gln/Tyr distal iron coordination, adaptation to extreme environmental conditions, and lack of a D helix suggests that ligand migration in HGbI differs from other previously studied globins. Locally enhanced molecular dynamics trajectories of oxygen migration indicate a large internal cavity. This may increase the tendency of oxygen to exit from portals other than the most direct exit from the space near the heme. Oxygen may reside transiently in shallow surface depressions around the exits. Such surface trapping may enhance both oxygen uptake by increasing contact time between molecules, and decrease release by increasing the probability of oxygen reentry from the vicinity of the portal.

Hell's Gate globin I: an acid and thermostable bacterial hemoglobin resembling mammalian neuroglobin.

Hell's Gate globin I (HGbI), a heme-containing protein structurally homologous to mammalian neuroglobins, has been identified from an acidophilic and thermophilic obligate methanotroph, Methylacidiphilum infernorum. HGbI has very high affinity for O(2) and shows barely detectable autoxidation in the pH range of 5.2-8.6 and temperature range of 25-50°C. Examination of the heme pocket by X-ray crystallography and molecular dynamics showed that conformational movements of Tyr29(B10) and Gln50(E7), as well as structural flexibility of the GH loop and H-helix, may play a role in modulating its ligand binding behavior. Bacterial HGbI's unique resistance to the sort of extreme acidity that would extract heme from any other hemoglobin makes it an ideal candidate for comparative structure-function studies of the expanding globin superfamily.

Globins synthesize the second messenger bis-(3'-5')-cyclic diguanosine monophosphate in bacteria.

Globin-coupled sensors are heme-binding signal transducers in Bacteria and Archaea in which an N-terminal globin controls the activity of a variable C-terminal domain. Here, we report that BpeGReg, a globin-coupled diguanylate cyclase from the whooping cough pathogen Bordetella pertussis, synthesizes the second messenger bis-(3'-5')-cyclic diguanosine monophosphate (c-di-GMP) upon oxygen binding. Expression of BpeGReg in Salmonella typhimurium enhances biofilm formation, while knockout of the BpeGReg gene of B. pertussis results in decreased biofilm formation. These results represent the first identification a signal ligand for any diguanylate cyclase and provide definitive experimental evidence that a globin-coupled sensor regulates c-di-GMP synthesis and biofilm formation. We propose that the synthesis of c-di-GMP by globin sensors is a widespread phenomenon in bacteria.

Derivatives of human complement component C3 for therapeutic complement depletion: a novel class of therapeutic agents.

To obtain proteins with the complement-depleting activity of Cobra Venom Factor (CVF), but with less immunogenicity, we have prepared human C3/CVF hybrid proteins, in which the C-terminus of the alpha-chain of human C3 is exchanged with homologous regions of the C-terminus of the beta-chain of CVF. We show that these hybrid proteins are able to deplete complement, both in vitro and in vivo. One hybrid protein, HC3-1496, is shown to be effective in reducing complement-mediated damage in two disease models in mice, collagen-induced arthritis and myocardial ischemia/reperfusion injury. Human C3/CVF hybrid proteins represent a novel class ofbiologicals as potential therapeutic agents in many diseases where complement is involved in the pathogenesis.

Docking of noncompetitive inhibitors into dengue virus type 2 protease: understanding the interactions with allosteric binding sites.

A group of flavanones and their chalcones, isolated from Boesenbergia rotunda L., were previously reported to show varying degrees of noncompetitive inhibitory activities toward Dengue virus type 2 (Den2) protease. Results obtained from automated docking studies are in agreement with experimental data in which the ligands were shown to bind to sites other than the active site of the protease. The calculated K(i) values are very small, indicating that the ligands bind quite well to the allosteric binding site. Greater inhibition by pinostrobin, compared to the other compounds, can be explained by H-bonding interaction with the backbone carbonyl of Lys74, which is bonded to Asp75 (one of the catalytic triad residues). In addition, structure-activity relationship analysis yields structural information that may be useful for designing more effective therapeutic drugs against dengue virus infections.

Ancestral hemoglobins in Archaea.

Hemoglobins are ubiquitous in Eukarya and Bacteria but, until now, have not been found in Archaea. A phylogenetic analysis of the recently revealed microbial family of globin-coupled heme-based sensors suggests that these sensors descended from an ancient globin-only progenitor, or a protoglobin (Pgb). Here, we report the discovery and characterization of two Pgbs from the Archaea: ApPgb from the obligately aerobic hyperthermophile Aeropyrum pernix, and MaPgb from the strictly anaerobic methanogen Methanosarcina acetivorans. Both ApPgb and MaPgb bind molecular oxygen, nitric oxide, and carbon monoxide by means of a heme moiety that is coordinated to the protein through the F8 histidine (histidine 120). We postulate that these archaeal globins are the ancestors of contemporary hemoglobins.