In Pseudomonas aeruginosa, Ttg2D is the soluble periplasmic phospholipid-binding component of an ABC transport system thought to be involved in maintaining the asymmetry of the outer membrane. Here ...we use the crystallographic structure of Ttg2D at 2.5 Å resolution to reveal that this protein can accommodate four acyl chains. Analysis of the available structures of Ttg2D orthologs shows that they conform a new substrate-binding-protein structural cluster. Native and denaturing mass spectrometry experiments confirm that Ttg2D, produced both heterologously and homologously and isolated from the periplasm, can carry two diacyl glycerophospholipids as well as one cardiolipin. Binding is notably promiscuous, allowing the transport of various molecular species. In vitro binding assays coupled to native mass spectrometry show that binding of cardiolipin is spontaneous. Gene knockout experiments in P. aeruginosa multidrug-resistant strains reveal that the Ttg2 system is involved in low-level intrinsic resistance against certain antibiotics that use a lipid-mediated pathway to permeate through membranes.
Increasing rates of antimicrobial resistance among uropathogens led, among other efforts, to the application of subtractive reverse vaccinology for the identification of antigens present in ...extraintestinal pathogenic E. coli (ExPEC) strains but absent or variable in non-pathogenic strains, in a quest for a broadly protective Escherichia coli vaccine. The protein coded by locus c5321 from CFT073 E. coli was identified as one of nine potential vaccine candidates against ExPEC and was able to confer protection with an efficacy of 33% in a mouse model of sepsis. c5321 (known also as EsiB) lacks functional annotation and structurally belongs to the Sel1-like repeat (SLR) family. Herein, as part of the general characterization of this potential antigen, we have focused on its structural properties.
We report the 1.74 Å-resolution crystal structure of c5321 from CFT073 E. coli determined by Se-Met SAD phasing. The structure is composed of 11 SLR units in a topological organisation that highly resembles that found in HcpC from Helicobacter pylori, with the main difference residing in how the super-helical fold is stabilised. The stabilising effect of disulfide bridges in HcpC is replaced in c5321 by a strengthening of the inter-repeat hydrophobic core. A metal-ion binding site, uncharacteristic of SLR proteins, is detected between SLR units 3 and 4 in the region of the inter-repeat hydrophobic core. Crystal contacts are observed between the C-terminal tail of one molecule and the C-terminal amphipathic groove of a neighbouring one, resembling interactions between ligand and proteins containing tetratricopeptide-like repeats.
The structure of antigen c5321 presents a mode of stabilization of the SLR fold different from that observed in close homologs of known structure. The location of the metal-ion binding site and the observed crystal contacts suggest a potential role in regulation of conformational flexibility and interaction with yet unidentified target proteins, respectively. These findings open new perspectives in both antigen design and for the identification of a functional role for this protective antigen.
Archaeal tetraether membrane lipids span the whole membrane width and present two C40 isoprenoid chains bound by two glycerol groups (or one glycerol and calditol). These lipids confer stability and ...maintain the membrane fluidity in mesophile to extremophile environments, making them very attractive for biotechnological applications. The isoprenoid lipid composition in archaeal membranes varies with temperature, which has placed these lipids in the focus of paleo-climatological studies for over a decade. Non-hydroxylated isoprenoid archaeal lipids are typically used as paleo-thermometry proxies, but recently identified hydroxylated (OH) derivatives have also been proposed as temperature proxies. The relative abundance of hydroxylated lipids increases at lower temperatures, but the physiological function of the OH moiety remains unknown. Here we present molecular dynamics simulations of membranes formed by the acyclic glycerol-dialkyl-glycerol-tetraether caldarchaeol (GDGT-0), the most widespread archaeal core lipid, and its mono-hydroxylated variant (OH-GDGT-0) to better understand the physico-chemical properties conferred to the membrane by this additional moiety. The molecular dynamics simulations indicate that the additional OH group forms hydrogen bonds mainly with the sugar moieties of neighbouring lipids and with water molecules, effectively increasing the size of the polar headgroups. The hydroxylation also introduces local disorder that propagates along the entire alkyl chains, resulting in a slightly more fluid membrane. These changes would help to maintain trans-membrane transport in cold environments, explaining why the relative abundance of hydroxylated Archaea lipids increases at lower temperatures. The in silico approach aids to understand the underlying physiological mechanisms behind the hydroxylated lipid based paleo-thermometer recently proposed.
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•We studied the effects of hydroxylation of GDGT-0 lipids by molecular dynamics.•OH moieties bulge out from the core lipid and extend the polar head group region.•The OH addition introduces local disorder that propagates along the core lipid.•Free cavities are displaced towards the inner part of the membrane.•Permeation of small solutes would be facilitated at low temperature.
How the solvent modulates the weak inter‐particle interactions in solution and affects macromolecule solubility is not yet understood. Well‐established thermodynamic relationships link second virial ...coefficient and preferential solute binding parameter. We present the meaning of these thermodynamic parameters and the way to measure them. When a solvation shell has a composition different from the bulk solvent, a negative contribution is found in the second virial coefficient corresponding to an effective attraction between the macromolecules in solution. A quantitative evaluation using simple models of solvated particles in solution suggests that solvation could induce, at high or low concentration of a small molecule solute, attractive inter‐particle interactions corresponding to favorable crystallization conditions.
We have investigated the potential of sedimentation velocity analytical ultracentrifugation for the measurement of the second virial coefficients of proteins, with the goal of developing a method ...that allows efficient screening of different solvent conditions. This may be useful for the study of protein crystallization. Macromolecular concentration distributions were modeled using the Lamm equation with the approximation of linear concentration dependencies of the diffusion constant,
D
=
D° (1
+
k
D
c), and the reciprocal sedimentation coefficient
s
=
s°/(1
+
k
s
c). We have studied model distributions for their information content with respect to the particle and its non-ideal behavior, developed a strategy for their analysis by direct boundary modeling, and applied it to data from sedimentation velocity experiments on halophilic malate dehydrogenase in complex aqueous solvents containing sodium chloride and 2-methyl-2,4-pentanediol, including conditions near phase separation. Using global modeling for three sets of data obtained at three different protein concentrations, very good estimates for
k
s and
s° and also for
D° and the buoyant molar mass were obtained. It was also possible to obtain good estimates for
k
D and the second virial coefficients. Modeling of sedimentation velocity profiles with the non-ideal Lamm equation appears as a good technique to investigate weak inter-particle interactions in complex solvents and also to extrapolate the ideal behavior of the particle.
DNA gyrase is the topoisomerase uniquely able to actively introduce negative supercoils into DNA. Vital in all bacteria, but absent in humans, this enzyme is a successful target for antibacterial ...drugs. From biophysical experiments in solution, we report the low-resolution structure of the full-length A subunit (GyrA). Analytical ultracentrifugation shows that GyrA is dimeric, but nonglobular. Ab initio modeling from small-angle X-ray scattering allows us to retrieve the molecular envelope of GyrA and thereby the organization of its domains. The available crystallographic structure of the amino-terminal domain (GyrA59) forms a dimeric core, and two additional pear-shaped densities closely flank it in an unexpected position. Each accommodates very well a carboxyl-terminal domain (GyrA-CTD) built from a homologous crystallographic structure. The uniqueness of gyrase is due to the ability of the GyrA-CTDs to wrap DNA. Their position within the GyrA structure strongly suggests a large conformation change of the enzyme upon DNA binding.
DNA gyrase, the only topoisomerase able to introduce negative supercoils into DNA, is essential for bacterial transcription and replication; absent from humans, it is a successful target for ...antibacterials. From biophysical experiments in solution, we report a structural model at ∼12–15 Å resolution of the full-length B subunit (GyrB). Analytical ultracentrifugation shows that GyrB is mainly a nonglobular monomer. Ab initio modeling of small-angle X-ray scattering data for GyrB consistently yields a “tadpole”-like envelope. It allows us to propose an organization of GyrB into three domains—ATPase, Toprim, and Tail—based on their crystallographic and modeled structures. Our study reveals the modular organization of GyrB and points out its potential flexibility, needed during the gyrase catalytic cycle. It provides important insights into the supercoiling mechanism by gyrase and suggests new lines of research.
Malate dehydrogenase (Hm MalDH) from the extreme halophile Haloarcula marismortui is a very acidic protein with extensive ion binding properties. It is a good model for the study of ...solvation−solubility relationships. We measured the small-angle neutron or X-ray scattering profiles of folded and stable Hm MalDH at various protein concentrations and derived the second virial coefficients A 2. In NaCl, CsCl, KF, KCl, and NaCH3CO2, A 2 values are positive, indicating globally repulsive protein−protein interactions. Below 1 M MgCl2 and MgSO4 or above 2 M (NH4)2SO4, A 2 rapidly decreases. From structure factor modeling with DLVO (Derjaguin, Landau, Verwey, and Overbeek)-like potentials, an effective diameter of 80−82 Å is found for the protein particle in solution, compatible with its structural dimensions; the effective charge of the particle is undefined because of the high salt concentration. The strong variations of the protein−protein interaction are correlated to an attractive potential whose depth evolves with the salinity but in an opposite way in Mg salts and (NH4)2SO4. A repulsive Donnan term, corresponding to counterion dissociation, and an attractive term related to previously measured preferential salt binding parameters are discussed from well-established thermodynamics considerations and qualitatively account for the behavior of the protein−protein interactions in the various solutions. Because a solvation shell with a composition different from bulk induces protein−protein attraction, molecular adaptation to high salt would be directed to allow protein−salt interactions in order to avoid water or salt enrichment at the surface of the protein and thus preserve its solubility.
Malate dehydrogenase from the extreme halophilic Haloarcula marismortui (Hm MalDH) is an acidic protein that is unstable below molar salt concentrations. The solvated folded protein was studied by ...small-angle neutron scattering in solvents containing salt: NaCl, NaCH3CO2, KF, NH4Cl, NH4CH3CO2, (NH4)2SO4, MgCl2, and MgSO4. It was found that the global solvent interactions depend mainly on the nature of the cation. Complementary mass density measurements in MgCl2, NaCl, NaCH3CO2, and (NH4)2SO4 allowed determining the partial molal volumes of the protein, which were found to increase slightly with the salt, and the preferential salt binding parameters for each solvent condition. These are strongly dependent on the cation type and salt concentration. Hm MalDH can be modeled as an invariant particle binding 4100 water molecules in MgCl2 and 2000 ± 200 in NaCl, NaCH3CO2, or (NH4)2SO4. The number of salt molecules associated to the particle decreases from about 85 to 0 in the order MgCl2 > NaCl = NaCH3CO2 > (NH4)2SO4. Alternatively, we considered exchangeable sites for water and salt with the effects of solvent nonideality. It does not change the description of the solvent interactions. Solvent anions act on Hm MalDH stability through a limited number of strong binding sites, as those seen at the interfaces of Hm MalDH by crystallography. Cations would act through some strong and numerous weak binding sites defined on the folded protein, in possible addition to nonspecific hydration effects.
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