Kanamycin-Mediated Conformational Dynamics of Escherichia coli Outer Membrane Protein TolC

TolC is a member of the outer membrane efflux proteins (OEPs) family and acts as an exit duct to export proteins, antibiotics, and substrate molecules across the Escherichia coli cell membrane. Export of these molecules is evidenced to be brought about through the reversible interactions and binding of substrate-specific drug molecules or antibiotics with TolC and by being open for transport, which afterward leads to cross-resistance. Hence, the binding of kanamycin with TolC was monitored through molecular docking (MD), the structural fluctuations and conformational changes to the atomic level. The results were further supported from the steady-state fluorescence binding and isothermal titration calorimetry (ITC) studies. Binding of kanamycin with TolC resulted in a concentration dependent fluorescence intensity quenching with 7 nm blue shift. ITC binding data maintains a single binding site endothermic energetic curve with binding parameters indicating an entropy driven binding process. The confirmational changes resulting from this binding were monitored by a circular dichroism (CD) study, and the results showed insignificant changes in the α-helix and β-sheets secondary structure contents, but the tertiary structure shows inclusive changes in the presence of kanamycin. The experimental data substaintially correlates the RMSD, Rg, and RMSF results. The resulting conformational changes of the TolC-kanamycin complexation was stabilized through H-bonding and other interactions.

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DENATURATION OF HUMAN SERUM ALBUMIN BY CERIUM (III) CHLORIDE

Biophysical Reviews and Letters ◽

10.1142/s1793048013500033 ◽

2013 ◽

Vol 08 (01n02) ◽

pp. 59-71

Author(s):

G. REZAEI BEHBAHANI ◽

M. SHALBAFAN ◽

N. GHEIBI ◽

L. BARZEGAR ◽

H. REZAEI BEHBAHANI ◽

...

Keyword(s):

Human Serum Albumin ◽

Serum Albumin ◽

Human Serum ◽

Conformational Changes ◽

Tertiary Structure ◽

Fluorescence Emission ◽

Titration Calorimetry ◽

Tryptophan Residue ◽

Spectroscopic Methods ◽

Irreversible Denaturation

Cerium (III) Chloride-induced conformational changes of human serum albumin, HSA, in phosphate buffer, 10 mM at pH 7.4 was investigated, using isothermal titration calorimetry (ITC), UV and fluorescence emission spectroscopic methods. The results indicate that CeCl3, Ce3+, induces irreversible denaturation of the HSA structure. The UV absorption intensity of HSA + Ce3+ shows a slight blueshift in the absorbance wavelength with increasing Ce3+ concentration. The fluorescence intensity was increased regularly and a slight redshift was observed in the emission wavelength. The HSA + Ce3+ complex quenches the fluorescence of HSA and changes the microenvironment of tryptophan residue. The emission intensity increases suggesting the loss of the tertiary structure of HSA. The results obtained from the ITC data are in agreement with the spectroscopic methods. The strong negative cooperativity of Ce3+ binding with HSA (Table 1) recovered from the extended solvation model, indicates that HSA has been denatured as a result of its interaction with Ce3+ ions.

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Inter-domain dynamics in the chaperone SurA and multi-site binding to its unfolded outer membrane protein clients

10.1101/2019.12.19.882696 ◽

2019 ◽

Author(s):

Antonio N. Calabrese ◽

Bob Schiffrin ◽

Matthew Watson ◽

Theodoros K. Karamanos ◽

Martin Walko ◽

...

Keyword(s):

Membrane Protein ◽

Outer Membrane ◽

Single Molecule ◽

Conformational Changes ◽

Outer Membrane Protein ◽

Conformational Dynamics ◽

Core Domain ◽

The Core ◽

Single Molecule Fret ◽

Dynamics Simulations

AbstractThe periplasmic chaperone SurA plays a key role in outer membrane protein (OMP) biogenesis. E. coli SurA comprises a core domain and two peptidylprolyl isomerase domains (P1 and P2), but how it binds its OMP clients and the mechanism(s) of its chaperone action remain unclear. Here, we have used chemical cross-linking, hydrogen-deuterium exchange, single-molecule FRET and molecular dynamics simulations to map the client binding site(s) on SurA and to interrogate the role of conformational dynamics of the chaperone’s domains in OMP recognition. We demonstrate that SurA samples a broad array of conformations in solution in which P2 primarily lies closer to the core/P1 domains than suggested by its crystal structure. Multiple binding sites for OMPs are located primarily in the core domain, with binding of the unfolded OMP resulting in conformational changes between the core/P1 domains. Together, the results portray a model in which unfolded OMP substrates bind in a cradle formed between the SurA domains, with structural flexibility between its domains assisting OMP recognition, binding and release.

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The Activity of Escherichia coli Chaperone SurA Is Regulated by Conformational Changes Involving a Parvulin Domain

Journal of Bacteriology ◽

10.1128/jb.00889-15 ◽

2016 ◽

Vol 198 (6) ◽

pp. 921-929 ◽

Cited By ~ 22

Author(s):

Garner R. Soltes ◽

Jaclyn Schwalm ◽

Dante P. Ricci ◽

Thomas J. Silhavy

Keyword(s):

Escherichia Coli ◽

Outer Membrane ◽

Conformational Changes ◽

Outer Membrane Proteins ◽

Membrane Integrity ◽

Minor Importance ◽

Type I ◽

Peptidyl Prolyl Isomerase ◽

Content Type ◽

Type I Fimbriae

ABSTRACTThe periplasmic chaperone SurA is critical for the biogenesis of outer membrane proteins (OMPs) and, thus, the maintenance of membrane integrity inEscherichia coli. The activity of this modular chaperone has been attributed to a core chaperone module, with only minor importance assigned to the two SurA peptidyl-prolyl isomerase (PPIase) domains. In this work, we used synthetic phenotypes and covalent tethering to demonstrate that the activity of SurA is regulated by its PPIase domains and, furthermore, that its activity is correlated with the conformational state of the chaperone. When combined with mutations in the β-barrel assembly machine (BAM), SurA mutations resulting in deletion of the second parvulin domain (P2) inhibit OMP assembly, suggesting that P2 is involved in the regulation of SurA. The first parvulin domain (P1) potentiates this autoinhibition, as mutations that covalently tether the P1 domain to the core chaperone module severely impair OMP assembly. Furthermore, these inhibitory mutations negate the suppression of and biochemically stabilize the protein specified by a well-characterized gain-of-function mutation in P1, demonstrating that SurA cycles between distinct conformational and functional states during the OMP assembly process.IMPORTANCEThis work reveals the reversible autoinhibition of the SurA chaperone imposed by a heretofore underappreciated parvulin domain. Many β-barrel-associated outer membrane (OM) virulence factors, including the P-pilus and type I fimbriae, rely on SurA for proper assembly; thus, a mechanistic understanding of SurA function and inhibition may facilitate antibiotic intervention against Gram-negative pathogens, such as uropathogenicEscherichia coli,E. coliO157:H7,Shigella, andSalmonella. In addition, SurA is important for the assembly of critical OM biogenesis factors, such as the lipopolysaccharide (LPS) transport machine, suggesting that specific targeting of SurA may provide a useful means to subvert the OM barrier.

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Determination of the native form of FadD, the Escherichia coli fatty acyl-CoA synthetase, and characterization of limited proteolysis by outer membrane protease OmpT

Biochemical Journal ◽

10.1042/bj3600699 ◽

2001 ◽

Vol 360 (3) ◽

pp. 699-706 ◽

Cited By ~ 2

Author(s):

Jae-Ho YOO ◽

Oscar H. CHENG ◽

Gerhard E. GERBER

Keyword(s):

Escherichia Coli ◽

Outer Membrane ◽

Conformational Changes ◽

Limited Proteolysis ◽

Fatty Acyl ◽

Native Form ◽

Terminal Fragment ◽

Sequencing And Expression

Several studies have described FadD, the Escherichia coli fatty acyl-CoA synthetase [also known as fatty acid:CoA ligase (AMP-forming); EC 6.2.1.3], as a 42–50kDa enzyme. Based on sequencing and expression data from the fadD gene, other reports have suggested that FadD is a 62kDa protein and represents the sole fatty acyl-CoA synthetase in E. coli. We report that the 62kDa FadD enzyme is a substrate for the outer membrane protease OmpT in vitro, producing a 43kDa C-terminal fragment and a 19kDa N-terminal fragment. Immunoblotting with a FadD antibody revealed that only the 62kDa form of the enzyme is present in vivo, but we utilized the proteolytic sensitivity of FadD to investigate its structure. Photoaffinity labelling experiments revealed that both intact FadD and the 43kDa fragment bound a long-chain fatty acid. Intact and cleaved FadD were also purified to determine the effect of cleavage on function. When using oleate as a substrate, cleaved FadD displayed 2-fold higher Km and Vmax values compared with intact FadD, but the catalytic efficiencies (kcat/Km) of the two forms were similar. This indicated that cleavage did not adversely affect enzyme activity. Proteolysis of FadD by OmpT was altered by the presence of oleate or ATP, both of which are ligands for the fatty acyl-CoA synthetase. This suggested that FadD undergoes ligand-induced conformational changes and implies that the region surrounding the cleavage site is mobile, a common characteristic of linker domains.

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Mosaic Evolution of Beta Barrel Porin Encoding Genes in Escherichia coli

10.1101/2021.09.21.461324 ◽

2021 ◽

Author(s):

Xiongbin Chen ◽

Xuxia Cai ◽

Zewei Chen ◽

Jinjin Wu ◽

Gaofeng Hao ◽

...

Keyword(s):

Escherichia Coli ◽

Membrane Proteins ◽

Outer Membrane ◽

Tertiary Structure ◽

Outer Membrane Proteins ◽

Common Mechanism ◽

General Strategy ◽

Variable Regions ◽

Mosaic Evolution ◽

Encoding Genes

AbstractBacterial porins serve as the interface interacting with extracellular environment, and are often found under positive selection to fit in different environmental stresses. Local recombination has been identified in a handful of porin genes to facilitate the rapid adaptation of bacterial cells. It remains unknown whether it is a common evolutionary mechanism in gram-negative bacteria for all or a majority of the outer membrane proteins. In this research, we investigated the β-barrel porin encoding genes in Escherichia coli that were reported under positive Darwinia selection. Besides fhuA that was found with ingenic local recombination predominantly previously, we identified four other genes, i.e., lamB, ompA, ompC and ompF, all showing the similar mosaic evolution patterns as in fhuA. Comparative analysis of the protein sequences disclosed a list of highly variable regions in each protein family, which are mostly located in the convex of extracellular loops and coinciding with the binding sites of various bacteriophages. For each of the porin family, mosaic recombination leads to various combinations of the HVRs with different sequence patterns, generating diverse protein groups. Structure modeling further indicated the conserved global topology for various groups of each porin family, but the extracellular surface varies a lot that is formed by individual or combinatorial HVRs. The conservation of global tertiary structure ensures the channel activity while the wide diversity of HVRs may assist bacteria avoiding the invasion of phages, antibiotics or immune surveillance factors. In summary, the study identified multiple bacterial porin genes with mosaic evolution, a likely general strategy, by which outer membrane proteins could facilitate the host bacteria to both maintain normal life processes and evade the attack of unflavored environmental factors rapidly.ImportanceMicroevolution studies can disclose more elaborate evolutionary mechanisms of genes, appearing especially important for genes with multifaceted function such as those encoding outer membrane proteins. However, in most cases, the gene is considered as a whole unit and the evolutionary patterns are disclosed. In this research, we reported that multiple bacterial porin proteins follow mosaic evolution, with local ingenic recombination combined with spontaneous mutations based positive Darwinia selection, and conservation for most of the other regions. It could represent a common mechanism for bacterial outer membrane proteins. The study also provides insights on development of new anti-bacterial agent or vaccines.

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Constant pH Molecular Dynamics Reveals How Proton Release Drives the Conformational Transition of a Transmembrane Efflux Pump

10.26434/chemrxiv.5496907 ◽

2017 ◽

Author(s):

Jana Shen ◽

Zhi Yue ◽

Helen Zgurskaya ◽

Wei Chen

Keyword(s):

Molecular Dynamics ◽

Conformational Changes ◽

Transmembrane Domain ◽

Conformational Transition ◽

Conformational Dynamics ◽

Proton Release ◽

Intrinsic Resistance ◽

Constant Ph ◽

Wide Range ◽

Constant Ph Molecular Dynamics

AcrB is the inner-membrane transporter of E. coli AcrAB-TolC tripartite efflux complex, which plays a major role in the intrinsic resistance to clinically important antibiotics. AcrB pumps a wide range of toxic substrates by utilizing the proton gradient between periplasm and cytoplasm. Crystal structures of AcrB revealed three distinct conformational states of the transport cycle, substrate access, binding and extrusion, or loose (L), tight (T) and open (O) states. However, the specific residue(s) responsible for proton binding/release and the mechanism of proton-coupled conformational cycling remain controversial. Here we use the newly developed membrane hybrid-solvent continuous constant pH molecular dynamics technique to explore the protonation states and conformational dynamics of the transmembrane domain of AcrB. Simulations show that both Asp407 and Asp408 are deprotonated in the L/T states, while only Asp408 is protonated in the O state. Remarkably, release of a proton from Asp408 in the O state results in large conformational changes, such as the lateral and vertical movement of transmembrane helices as well as the salt-bridge formation between Asp408 and Lys940 and other sidechain rearrangements among essential residues.Consistent with the crystallographic differences between the O and L protomers, simulations offer dynamic details of how proton release drives the O-to-L transition in AcrB and address the controversy regarding the proton/drug stoichiometry. This work offers a significant step towards characterizing the complete cycle of proton-coupled drug transport in AcrB and further validates the membrane hybrid-solvent CpHMD technique for studies of proton-coupled transmembrane proteins which are currently poorly understood.

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Molecular Basis of Glucose Transport Mechanism in Plants

10.26434/chemrxiv.8049848.v1 ◽

2019 ◽

Cited By ~ 2

Author(s):

Balaji Selvam ◽

Ya-Chi Yu ◽

Liqing Chen ◽

Diwakar Shukla

Keyword(s):

Molecular Dynamics ◽

Free Energy ◽

Molecular Dynamics Simulations ◽

Conformational Changes ◽

Transport Mechanism ◽

Conformational Dynamics ◽

Site Directed Mutagenesis ◽

Free Energy Barrier ◽

Transport Cycle ◽

Dynamics Simulations

The SWEET family belongs to a class of transporters in plants that undergoes large conformational changes to facilitate transport of sugar molecules across the cell membrane. However, the structures of their functionally relevant conformational states in the transport cycle have not been reported. In this study, we have characterized the conformational dynamics and complete transport cycle of glucose in OsSWEET2b transporter using extensive molecular dynamics simulations. Using Markov state models, we estimated the free energy barrier associated with different states as well as 1 for the glucose the transport mechanism. SWEETs undergoes structural transition to outward-facing (OF), Occluded (OC) and inward-facing (IF) and strongly support alternate access transport mechanism. The glucose diffuses freely from outside to inside the cell without causing major conformational changes which means that the conformations of glucose unbound and bound snapshots are exactly same for OF, OC and IF states. We identified a network of hydrophobic core residues at the center of the transporter that restricts the glucose entry to the cytoplasmic side and act as an intracellular hydrophobic gate. The mechanistic predictions from molecular dynamics simulations are validated using site-directed mutagenesis experiments. Our simulation also revealed hourglass like intermediate states making the pore radius narrower at the center. This work provides new fundamental insights into how substrate-transporter interactions actively change the free energy landscape of the transport cycle to facilitate enhanced transport activity.

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