Structural Basis for Enzyme I Inhibition by α‑Ketoglutarate

Creating new bacterial strains in which carbon and nitrogen metabolism are uncoupled is potentially very useful for optimizing yields of microbial produced chemicals from renewable carbon sources. However, the mechanisms that balance carbon and nitrogen consumption in bacteria are poorly understood. Recently, α-ketoglutarate (αKG), the carbon substrate for ammonia assimilation, has been observed to inhibit <i>Escherichia coli</i> enzyme I (EI), the first component of the bacterial phosphotransferase system (PTS), thereby providing a direct biochemical link between central carbon and nitrogen metabolism. Here we investigate the EI-αKG interaction by NMR and enzymatic assays. We show that αKG binds with a <i>K</i>_D of ∼2.2 mM at the active site of EI, acting as a competitive inhibitor. In addition, we use molecular docking simulations to derive a structural model of the enzyme–inhibitor complex that is fully consistent with NMR and analytical ultracentrifugation data. We expect that the EI-αKG structure presented here will provide a starting point for structure-based design of EI mutants resistant to αKG

Hydrodynamic and Functional Analysis of HIV-1 Vif Oligomerization

HIV-1 Vif is an accessory protein that induces the proteasomal degradation of the host restriction factor, apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G). The N-terminal half of Vif binds to APOBEC3G, and the C-terminal half binds to subunits of a cullin 5-based ubiquitin ligase. This Vif-directed ubiquitin ligase induces the degradation of APOBEC3G (a cytidine deaminase) and thereby protects the viral genome from mutation. A conserved PPLP motif near the C-terminus of Vif is essential for Vif function and is also involved in Vif oligomerization. However, the mechanism and functional significance of Vif oligomerization is unclear. We employed analytical ultracentrifugation to examine the oligomeric properties of Vif in solution. Contrary to previous reports, we find that Vif oligomerization does not require the conserved PPLP motif. Instead, our data suggest a more complex mechanism involving interactions among the HCCH motif, the BC box, and downstream residues in Vif. Mutation of residues near the PPLP motif (S165 and V166) affected the oligomeric properties of Vif and weakened the ability of Vif to bind and induce the degradation of APOBEC3G. We propose that Vif oligomerization may represent a mechanism for regulating interactions with APOBEC3G

Tilted, Uninterrupted, Monomeric HIV‑1 gp41 Transmembrane Helix from Residual Dipolar Couplings

Cryo-electron microscopy and X-ray crystallography have shown that the pre- and postfusion states of the HIV-1 gp41 viral coat protein, although very different from one another, each adopt <i>C</i>₃ symmetric structures. A stable homotrimeric structure for the transmembrane domain (TM) also was modeled and supported by experimental data. For a <i>C</i>₃ symmetric structure, alignment in an anisotropic medium must be axially symmetric, with the unique axis of the alignment tensor coinciding with the <i>C</i>₃ axis. However, NMR residual dipolar couplings (RDCs) measured under three different alignment conditions were found to be incompatible with <i>C</i>₃ symmetry. Subsequent measurements by paramagnetic relaxation enhancement, analytical ultracentrifugation, and DEER EPR, indicate that the transmembrane domain is monomeric. ¹⁵N NMR relaxation data and RDCs show that TM is highly ordered and uninterrupted for a total length of 32 residues, extending well into the membrane proximal external region

Amino acid sequence, domain organization and structure representations of HIV-1 gp41.

<p>(A) Numbering of residues corresponds to their positions in the HIV-1 Env sequence. Gp41 spans residues 512 to 856 (in red) of gp160. (B) Abbreviations are as follows: FP, fusion peptide; FPPR, fusion peptide proximal region; N-HR, N-heptad repeat; IL, immune-dominant linker; C-HR, C-heptad repeat; MPER, membrane proximal external region; TM, transmembrane region; and CT, intraviral C-terminal domain, respectively. For ease of designating the various constructs used in this study, gp41 is renumbered from 1–345 (in black). The longest sequence used in this study spans residues 17 to 194 corresponding to Env numbering 528 to 705. Except for 35-144^IL, all constructs have their IL region (residues 69 through 116) replaced with the L6 spacer (SGGRGG) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160597#pone.0160597.s001" target="_blank">S1 Fig</a>). They are designated based on the amino acid sequence they encompass. When not substituted with the L6 spacer, the construct is designated with an IL (in superscript) following the designation. The various constructs used are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160597#pone.0160597.s001" target="_blank">S1 Fig</a> (C). The 6HB spanning A533 to R580 (FPPR/N-HR) and M629-N677 (C-HR/MPER) is modeled from pdb entries 1SZT [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160597#pone.0160597.ref014" target="_blank">14</a>] and 2X7R [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160597#pone.0160597.ref017" target="_blank">17</a>]. Terminal residues are indicated in red on the ribbon. Positions substituted with cysteines for MTSL-labeling (chemistry shown in D) are R633, L661, G527 and L684. These positions are indicated also in red on the ribbon (in C) and the sequence with a ball and stick (in A). The epitopes recognized by the antibodies 2F5, 4E10 and Z13e1 are shown in (A).</p

Insights into the Conformation of the Membrane Proximal Regions Critical to the Trimerization of the HIV-1 gp41 Ectodomain Bound to Dodecyl Phosphocholine Micelles

<div><p>The transitioning of the ectodomain of gp41 from a pre-hairpin to a six-helix bundle conformation is a crucial aspect of virus-cell fusion. To gain insight into the intermediary steps of the fusion process we have studied the pH and dodecyl phosphocholine (DPC) micelle dependent trimer association of gp41 by systematic deletion analysis of an optimized construct termed 17–172 (residues 528 to 683 of Env) that spans the fusion peptide proximal region (FPPR) to the membrane proximal external region (MPER) of gp41, by sedimentation velocity and double electron-electron resonance (DEER) EPR spectroscopy. Trimerization at pH 7 requires the presence of both the FPPR and MPER regions. However, at pH 4, the protein completely dissociates to monomers. DEER measurements reveal a partial fraying of the C-terminal MPER residues in the 17–172 trimer while the other regions, including the FPPR, remain compact. In accordance, truncating nine C-terminal MPER residues (675–683) in the 17–172 construct does not shift the trimer-monomer equilibrium significantly. Thus, in the context of the gp41 ectodomain spanning residues 17–172, trimerization is clearly dependent on FPPR and MPER regions even when the terminal residues of MPER unravel. The antibody Z13e1, which spans both the 2F5 and 4E10 epitopes in MPER, binds to 17–172 with a <i>K</i>_d of 1 ± 0.12 μM. Accordingly, individual antibodies 2F5 and 4E10 also recognize the 17–172 trimer/DPC complex. We propose that binding of the C-terminal residues of MPER to the surface of the DPC micelles models a correct positioning of the trimeric transmembrane domain anchored in the viral membrane.</p></div

Binding isotherm for the interaction of Z13e1 with 17–172.

<p>The peaks in red indicate the heat released after each addition of 17–172 into the antibody solution. Traces in blue indicate control titrations using either just the antibody in the cell titrated with the buffer (10 mM Tris-HCl, pH 7.6, 150 mM NaCl, 2 mM DPC) shown in B (black) or the same buffer titrated with the antigen shown in A and B (blue). (C) The data were best fit using a single binding constant to calculate the thermodynamic parameters.</p

DEER EPR measurements with MTSL labels in various positions of the 17–172 construct.

<p>Positions of labels are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160597#pone.0160597.g001" target="_blank">Fig 1</a>. Results of the DeerAnalysis2015 Tikhonov Regularization fits [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160597#pone.0160597.ref048" target="_blank">48</a>] of the background corrected data acquired at pH 7 and pH 4 in the presence of ~2-fold excess of DPC micelles. The regularization parameter, α was determined by examination of the relevant L-curves (α = 10 in all cases except for the ^N-Cys17-172 data where α = 1000 was the best choice). Plots of the raw data are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160597#pone.0160597.s006" target="_blank">S6</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160597#pone.0160597.s007" target="_blank">S7</a> Figs. Previously published data indicating the fraying of the C-HR region by the addition of C34 to the 6HB formed by the construct 35–144 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160597#pone.0160597.s001" target="_blank">S1 Fig</a>) in 10 mM Tris-HCL, pH 7.6, and 150 mM NaCl (without DPC) is superimposed (orange, panel F) for comparison with 17-172^C-Cys in the presence of DPC at pH 4. The blue trace depicts the distance distribution of 35–144 trimer in the absence of C34 and DPC at pH 7. P(<i>r</i>) distribution calculated using the MMM software package for 17-172^L661C labeled with MTSL is superimposed for comparison with the experimental data in panel G (red trace).</p

Mass estimations by sedimentation velocity analysis.

<p>Mass estimations by sedimentation velocity analysis.</p

Sedimentation velocity absorbance <i>c(s)</i> distributions for MTSL-labeled 17–172.

<p>Experiments were carried out using 50 μM ²H-MTSL labeled, 17-172^L661C (blue) and 17-172^C-Cys (red) in 50 mM sodium acetate, pH 4, and 31% nondeuterated glycerol in the presence of ~2-fold excess of DPC micelles. Samples were prepared similar to those used for DEER at a concentration of 50 μM with the above buffer in H₂O instead of D₂O. 17–172 construct which is mainly a monomer in 50 mM sodium acetate at pH 4 and DPC, but in the absence of glycerol, is shown as control (black, rescaled to fit c(<i>s</i>) axis).</p

Sedimentation velocity absorbance <i>c(s)</i> distributions for various gp41 analogues at 20°C in the presence of an excess of DPC micelles.

<p>(A-C) Data collected in 20 mM sodium phosphate, pH 7 and 150 mM NaCl, except for 17–194 which was recorded in the absence of NaCl. Values derived for constructs A through I at pH 7 are listed numerically in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160597#pone.0160597.t001" target="_blank">Table 1</a>. SV data for 17–194 at pH 4.2 was acquired in 50 mM sodium acetate with 25 mM KCl and 10 mM DPC (panel A, blue) and 17–172 at pH 4 and 5 (panel D: green and orange, respectively) in 50 mM sodium acetate with 10 mM DPC. Protein concentrations are as follows: 17–194 (pH 4.2), ~10 μM; 17–194 (pH 7), 1–2 μM; all others, 10–20 μM. The trimer/monomer distribution was unchanged in the absence or presence of 150 mM NaCl at pH 6 (panel D, red dotted trace). Slight variations in the sedimentation coefficients for the 17–172 and 17–172 ^Cys-MTSL trimer arise from differences in the isotopic labeling: ²H ¹³C ¹⁵N-labeled 17–172 complexed with ¹H-DPC as compared to ²H-labeled 17-172^Cys-MTSL complexed with ²H-DPC.</p