Conformational changes and loose packing promote E. coli Tryptophanase cold lability

<p>Abstract</p> <p>Background</p> <p>Oligomeric enzymes can undergo a reversible loss of activity at low temperatures. One such enzyme is tryptophanase (Trpase) from <it>Escherichia coli</it>. Trpase is a pyridoxal phosphate (PLP)-dependent tetrameric enzyme with a Mw of 210 kD. PLP is covalently bound through an enamine bond to Lys270 at the active site. The incubation of holo <it>E. coli </it>Trpases at 2°C for 20 h results in breaking this enamine bond and PLP release, as well as a reversible loss of activity and dissociation into dimers. This sequence of events is termed cold lability and its understanding bears relevance to protein stability and shelf life.</p> <p>Results</p> <p>We studied the reversible cold lability of <it>E. coli </it>Trpase and its Y74F, C298S and W330F mutants. In contrast to the holo <it>E. coli </it>Trpase all apo forms of Trpase dissociated into dimers already at 25°C and even further upon cooling to 2°C. The crystal structures of the two mutants, Y74F and C298S in their apo form were determined at 1.9Å resolution. These apo mutants were found in an open conformation compared to the closed conformation found for <it>P. vulgaris </it>in its holo form. This conformational change is further supported by a high pressure study.</p> <p>Conclusion</p> <p>We suggest that cold lability of <it>E. coli </it>Trpases is primarily affected by PLP release. The enhanced loss of activity of the three mutants is presumably due to the reduced size of the side chain of the amino acids. This prevents the tight assembly of the active tetramer, making it more susceptible to the cold driven changes in hydrophobic interactions which facilitate PLP release. The hydrophobic interactions along the non catalytic interface overshadow the effect of point mutations and may account for the differences in the dissociation of <it>E. coli </it>Trpase to dimers and <it>P. vulgaris </it>Trpase to monomers.</p

Functional Mimetics of the HIV-1 CCR5 Co-Receptor Displayed on the Surface of Magnetic Liposomes.

Chemokine G protein coupled receptors, principally CCR5 or CXCR4, function as co-receptors for HIV-1 entry into CD4+ T cells. Initial binding of the viral envelope glycoprotein (Env) gp120 subunit to the host CD4 receptor induces a cascade of structural conformational changes that lead to the formation of a high-affinity co-receptor-binding site on gp120. Interaction between gp120 and the co-receptor leads to the exposure of epitopes on the viral gp41 that mediates fusion between viral and cell membranes. Soluble CD4 (sCD4) mimetics can act as an activation-based inhibitor of HIV-1 entry in vitro, as it induces similar structural changes in gp120, leading to increased virus infectivity in the short term but to virus Env inactivation in the long term. Despite promising clinical implications, sCD4 displays low efficiency in vivo, and in multiple HIV strains, it does not inhibit viral infection. This has been attributed to the slow kinetics of the sCD4-induced HIV Env inactivation and to the failure to obtain sufficient sCD4 mimetic levels in the serum. Here we present uniquely structured CCR5 co-receptor mimetics. We hypothesized that such mimetics will enhance sCD4-induced HIV Env inactivation and inhibition of HIV entry. Co-receptor mimetics were derived from CCR5 gp120-binding epitopes and functionalized with a palmitoyl group, which mediated their display on the surface of lipid-coated magnetic beads. CCR5-peptidoliposome mimetics bound to soluble gp120 and inhibited HIV-1 infectivity in a sCD4-dependent manner. We concluded that CCR5-peptidoliposomes increase the efficiency of sCD4 to inhibit HIV infection by acting as bait for sCD4-primed virus, catalyzing the premature discharge of its fusion potential

Effect of co-display of CD4- and CCR5-peptidomimetics on the ability of CCR5-peptidoliposomes to inhibit HIV-1.

<p>R5-tropic JRFL-pseudotyped HIV-1 was co-incubated for 2 h with: CCR5-Beads–peptidoliposomes containing 5% each of NT-3FSN-CCR5-PAL and ECL2-CCR5-2PAL, or (CD4+CCR5)-Beads–peptidoliposomes containing 5% each of CD4M48-PAL, ECL2-CCR5-2PAL and NT-3FSN-CCR5-PAL. Peptide-free magnetic liposomes were used as control (set to 100% infectivity). The virus was separated from the beads by a magnetic field and TZM-HeLa-β-gal target cells were infected for 4 h. β-gal expression was carried out 48 h post infection. The data are mean ± S.E.M. calculated from three independent experiments each performed in duplicate.</p

CCR5-peptidoliposomes enhance the ability of soluble CD4 mimetic to inhibit infection of R5-tropic HIV-1.

<p>CCR5-peptidoliposomes were co-incubated for 2 h with R5-tropic JRFL-pseudotyped HIV-1 (A); R5-tropic ADA-pseudotyped HIV-1 (B); or X4-tropic HXB2-pseudotyped HIV-1 (C), in the presence or absence of different concentrations of sCD4M48. Peptide-free magnetic liposomes (in the absence of sCD4M48) were used as control (set to 100% infectivity). The virus was separated from the beads by a magnetic field and TZM-HeLa-β-gal target cells were infected for 4 h. β-gal expression was carried out 48 h post infection. An unpaired <i>t</i>-test (two-tailed) was used to assess the significance of the difference in the means observed between the two groups indicated, <i>p</i> < 0.05. The data are mean ± S.E.M. calculated from three independent experiments each performed in duplicate.</p

Magnetic liposome population is homogeneous.

<p>The magnetic liposomes were prepared using a lipid mixture POPC:POPE:DMPA (molar ratio 6:3:1) containing 1% Biotinyl-DOPE in the presence (black peak) or absence (white peak) of 1% Rhodamine–DOPE, and analyzed by FACS as described in the Materials and Methods. FL1-H designates the height of the photon peak obtained by using a 525/50 band pass filter (FL1). The figure shows the data for an experiment representative of two similar experiments.</p

CCR5-peptidoliposomes bind soluble recombinant gp120.

<p>Peptidoliposomes were incubated with 4 μM soluble recombinant His-tagged gp120 for 1 h at 37°C and the binding was assessed as described in the Materials and Methods. Mimetics used: mCD4 –CD4M48-PAL (1%); mCCR5-Nt – NT-2Y-CCR5-PAL (1%); mCCR5-ECL2 –ECL2-CCR5-2PAL (1%); mCCR5-Nt-3FSN–NT-3FSN-CCR5-PAL (1%); sCD4M48 –soluble M48 peptide (10 μM) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144043#pone.0144043.ref015" target="_blank">15</a>]. Control – peptide-free magnetic liposomes. <i>p</i> < 0.05. The data are mean ± S.E.M. calculated from three independent experiments each performed in triplicate.</p

Palmitoylated CCR5-peptidomimetics can be displayed on liposome surface.

<p>Peptidoliposomes were generated in the presence of increasing concentrations (molar %) of NT-2Y-CCR5-PAL (A) or ECL2-CCR5-2PAL (B) in the lipid mixture. (C) Peptidoliposomes were formed in the presence of 1% ECL2-CCR5-2PAL and the indicated concentrations of NT-2Y-CCR5-PAL in the lipid mixture. Peptidoliposomes were reacted with anti-CCR5 N-terminus polyclonal antibody (ab 7346, Abcam) (A) or with anti-CCR5 ECL2 (raised against a synthetic peptide 2D7-2SK [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144043#pone.0144043.ref044" target="_blank">44</a>]) polyclonal antibody (ab 36818, Abcam) (B and C), followed by a secondary HRP-conjugated antibody (ab 7090, Abcam) and analyzed as described in the Materials and Methods. Control – peptide-free magnetic liposomes. The data are mean ± S.E.M. calculated from three independent experiments each performed in triplicate.</p

HIV-1 incubation with CCR5-peptidoliposomes does not deplete viral loads.

<p>JRFL-pseudotyped HIV-1 was incubated for 2 h with peptidoliposomes in the presence or absence of M48 peptide–sCD4M48 (10 μM). CCR5-Beads - peptidoliposomes containing 5% each of NT-3FSN-CCR5-PAL and ECL2-CCR5-2PAL; (CD4+CCR5)-Beads - peptidoliposomes containing 5% each of CD4M48-PAL, ECL2-CCR5-2PAL and NT-3FSN-CCR5-PAL. At the end of the incubation, the supernatant and liposome fractions were analyzed by ELISA for the presence of HIV-p24 antigen as described in the Materials and Methods. Peptide-free magnetic liposomes were used as the control (p24 count in the supernatant set to 100%). The data are mean ± S.E.M. calculated from three independent experiments each performed in duplicate.</p