14 research outputs found
Unusual Constriction Zones in the Major Porins OmpU and OmpT from Vibrio cholerae
No full textThe outer membranes (OM) of many Gram-negative bacteria contain general porins, which form nonspecific, large-diameter channels for the diffusional uptake of small molecules required for cell growth and function. While the porins of Enterobacteriaceae (e.g., E. coli OmpF and OmpC) have been extensively characterized structurally and biochemically, much less is known about their counterparts in Vibrionaceae. Vibrio cholerae, the causative agent of cholera, has two major porins, OmpU and OmpT, for which no structural information is available despite their importance for the bacterium. Here we report high-resolution X-ray crystal structures of V. cholerae OmpU and OmpT complemented with molecular dynamics simulations. While similar overall to other general porins, the channels of OmpU and OmpT have unusual constrictions that create narrower barriers for small-molecule permeation and change the internal electric fields of the channels. Together with electrophysiological and in vitro transport data, our results illuminate small-molecule uptake within the Vibrionaceae. Pathania et al. describe the X-ray structures of the major Vibrio cholerae porins, OmpU and OmpT. The channels have narrow pore sizes and altered internal electric fields due to the presence of additional, unusual constriction elements. In addition, the interaction of deoxycholate and carbapenems with OmpU and OmpT are reported
Getting Drugs through Small Pores: Exploiting the Porins Pathway in Pseudomonas aeruginosa
No full textUnderstanding molecular properties of outer membrane channels of Gram-negative bacteria is of fundamental significance as they are the entry point of polar antibiotics into bacteria. Outer membrane proteomics revealed OccK8 (OprE) to be among the five most expressed substrate specific channels of the clinically important Pseudomonas aeruginosa. The high-resolution X-ray structure and electrophysiology highlighted a very narrow pore. However, experimental in vitro methods showed the transport of natural amino acids and antibiotics, among them ceftazidime. We used molecular dynamics simulations to reveal the importance of the physicochemical properties of ceftazidime in modulating the translocation through OccK8, proposing a structure–function relationship. As in general porins, the internal electric field favors the translocation of polar molecules by gainful energy compensation in the central constriction region. Importantly, the comparatively narrow OccK8 pore can undergo a substrate-induced expansion to accommodate relatively large-sized substrates
Characterisation of the major porins OmpU and OmpT of Vibrio cholerae
No full textPhD ThesisThe asymmetric outer membrane (OM) of a Gram-negative bacterium has many proteins embedded as β-barrel structures in it called outer membrane proteins (OMPs). The majority of these OMPs (porins) form non-selective channels across the OM to allow passive uptake of substrates. The treatment for infections caused by such bacteria mostly involves the administration of drugs/antibiotics, for which these porins play a very crucial role by providing an efficient (although not yet fully understood) route through their channel. The goal of this study is to study small-molecule permeation through the major porins, OmpU and OmpT, of Vibrio cholerae (the causative agent of cholera) for potential use of these proteins as the target for designing antibiotics or vaccines. Towards this project, we have succeeded in solving the 3D X-ray crystal structures of OmpU and OmpT as well as the structures of the major porins from Klebsiella pneumoniae (OmpK36) and Enterobacter cloacae (OmpE36, OmpE35).
The proteins (OmpU/T, OmpE35/E36 and OmpK36) show the typical arrangement of porins with three β-barrel monomers arranged into a trimer. Each monomer displays 16 antiparallel β-strands forming the hollow β-barrel formed by 8 long extracellular loops and 8 short periplasmic turns. The latching loop L2 stabilises the trimer while loop L3 departs from the β-barrel fold and constricts the pore half-way through the channel. An unusual feature is observed in the channels of OmpU and OmpT that distinguishes them from other typical porins. In OmpU, the first 10 residues of N-terminus insert into the barrel and constrict the pore. In contrast, the structure of OmpT reveals that the extracellular loop L8 folds inwards to constrict the lumen of the channel. Such constriction elements not only reduce the pore sizes of OmpU and OmpT but may also dramatically affect the internal electrostatics of these channels, which is very important for small-molecule permeation. In addition, we also performed single channel electrophysiology experiments with OmpU and OmpT which revealed interesting features with the addition of carbapenems.European Union’s Seventh Framework Programme (FP7/2007–2013) and European Federation of Pharmaceutical Industries and Associations companies in kind contribution. Therefore, a very special gratitude goes out to all down to EU Marie Curie network (ITN) for funding my PhD
How to get large drugs through small pores? Exploiting the porins pathway in Pseudomonas aeruginosa
No full textAxisymmetric deformation in a micropolar thermoelastic medium under fractional order theory of thermoelasticity
No full textQuantum chemical investigations on the molecular structure, FTIR, UV-Vis and HOMO-LUMO analysis of 15-16-epoxy-7b, 9a dihydroxylabdane 13(16), 14-dien-6-one
No full textGetting Drugs into Gram-Negative Bacteria: Rational Rules for Permeation through General Porins
No full textSmall, hydrophilic molecules, including most important antibiotics in clinical use, cross the Gram-negative outer membrane through the water-filled channels provided by porins. We have determined the X-ray crystal structures of the principal general porins from three species of Enterobacteriaceae, namely Enterobacter aerogenes, Enterobacter cloacae, and Klebsiella pneumoniae, and determined their antibiotic permeabilities as well as those of the orthologues from Escherichia coli. Starting from the structure of the porins and molecules, we propose a physical mechanism underlying transport and condense it in a computationally efficient scoring function. The scoring function shows good agreement with in vitro penetration data and will enable the screening of virtual databases to identify molecules with optimal permeability through porins and help to guide the optimization of antibiotics with poor permeation
Structural basis for the inhibition of the Bacillus subtilis c-di-AMP cyclase CdaA by the phosphoglucomutase GlmM
No full textCyclic-di-adenosine monophosphate (c-di-AMP) is an important nucleotide signaling molecule that plays a key role in osmotic regulation in bacteria. c-di-AMP is produced from two molecules of ATP by proteins containing a diadenylate cyclase (DAC) domain. In Bacillus subtilis, the main c-di-AMP cyclase, CdaA, is a membrane-linked cyclase with an N-terminal transmembrane domain followed by the cytoplasmic DAC domain. As both high and low levels of c-di-AMP have a negative impact on bacterial growth, the cellular levels of this signaling nucleotide are tightly regulated. Here we investigated how the activity of the B. subtilis CdaA is regulated by the phosphoglucomutase GlmM, which has been shown to interact with the c-di-AMP cyclase. Using the soluble B. subtilis CdaACD catalytic domain and purified full-length GlmM or the GlmMF369 variant lacking the C-terminal flexible domain 4, we show that the cyclase and phosphoglucomutase form a stable complex in vitro and that GlmM is a potent cyclase inhibitor. We determined the crystal structure of the individual B. subtilis CdaACD and GlmM homodimers and of the CdaACD:GlmMF369 complex. In the complex structure, a CdaACD dimer is bound to a GlmMF369 dimer in such a manner that GlmM blocks the oligomerization of CdaACD and formation of active head-to-head cyclase oligomers, thus suggesting a mechanism by which GlmM acts as a cyclase inhibitor. As the amino acids at the CdaACD:GlmM interphase are conserved, we propose that the observed mechanism of inhibition of CdaA by GlmM may also be conserved among Firmicutes
NUCLEAR QUADRUPOLE RESONANCE ZEEMAN STUDY OF
No full textAuthor Institution: Department of Chemistry, Virginia Polytechnic Institute and State UniversityThe Zeeman splittings of the nuclear quadrupole resonance transition at 16.303 MHz in a single crystal of barium chloride dihydrate, has been studied and the zero-splitting loci for both physically non-equivalent lattice sites have been determined. From the study it was found that the nuclear quadrupole coupling constant is = 29.8 MHz and that the asymmetry parameter is . The direction of the electric field gradient (EFG) principal axis system relative to the crystal axis system was found. The magnitudes and directions of the EFG tensor components were calculated by using a point charge-point dipole model and isotropic polarizabilities. By using a projected Sternheimer factor of -80 this leads to a calculated coupling constant of 60 MHz and an asymmetry parameter of 0.1. Both the experimental and calculated values of the parameters are in agreement with the crystal symmetry. In view of the simplifications inherent in the model used this represents reasonable agreement
Modeling of Specific Lipopolysaccharide Binding Sites on a Gram-Negative Porin
No full textProtein-lipopolysaccharide
(LPS) interactions play an important
role in providing a stable outer membrane to Gram-negative bacteria.
However, the LPS molecules are highly viscous, and sampling LPS motions
is thus challenging on a microsecond time scale in simulations. To
this end, we introduce a new protocol to randomly allow the LPS molecules
to self-assemble around the protein and thereby reduce the starting
bias in the simulations. Here we present all-atom molecular dynamics
simulations of the OmpE36 porin in an outer membrane model which sum
up to a simulation time of more than 20 μs and identify the
geometrical properties of the first LPS shell and the role of calcium
ions in LPS binding to the protein. The simulations reproduce LPS
binding to the porin observed in a recently determined crystal structure
but not as compact as in the crystal structure. In addition, the influence
of the outer membrane environment on the protein dynamics was analyzed.
Our findings highlight the role of divalent cations in stabilizing
the binding between proteins and LPS molecules in the outer membrane
of Gram-negative bacteria
