Molecular Crowding Enhanced ATPase Activity of the RNA Helicase eIF4A Correlates with Compaction of Its Quaternary Structure and Association with eIF4G

Enzymatic reactions occur in a crowded and confined environment <i>in vivo,</i> containing proteins, RNA and DNA. Previous reports have shown that interactions between macromolecules, and reactions rates differ significantly between crowded environments and dilute buffers. However, the direct effect of crowding on the level of high-resolution structures of macromolecules has not been extensively analyzed and is not well understood. Here we analyze the effect of macromolecular crowding on structure and function of the human translation initiation factors eIF4A, a two-domain DEAD-Box helicase, the HEAT-1 domain of eIF4G, and their complex. We find that crowding enhances the ATPase activity of eIF4A, which correlates with a shift to a more compact structure as revealed with small-angle X-ray scattering. However, the individual domains of eIF4A, or the eIF4G-HEAT-1 domain alone show little structural changes due to crowding except for flexible regions. Thus, the effect of macromolecular crowding on activity and structure need to be taken into account when evaluating enzyme activities and structures of multidomain proteins, proteins with flexible regions, or protein complexes obtained by X-ray crystallography, NMR, or other structural methods

Controlled Co-reconstitution of Multiple Membrane Proteins in Lipid Bilayer Nanodiscs Using DNA as a Scaffold

Nanodiscs constitute a tool for the solubilization of membrane proteins in a lipid bilayer, thus offering a near-native membrane environment. Many membrane proteins interact with other membrane proteins; however, the co-reconstitution of multiple membrane proteins in a single nanodisc is a random process that is adversely affected by several factors, including protein aggregation. Here, we present an approach for the controlled co-reconstitution of multiple membrane proteins in a single nanodisc. The temporary attachment of designated oligonucleotides to individual membrane proteins enables the formation of stable, detergent-solubilized membrane protein complexes by base-pairing of complementary oligonucleotide sequences, thus facilitating the insertion of the membrane protein complex into nanodiscs with defined stoichiometry and composition. As a proof of principle, nanodiscs containing a heterodimeric and heterotrimeric membrane protein complex were reconstituted using a fluorescently labeled voltage-gated anion channel (VDAC) as a model system

Identification of a Conserved LDFLP Sequence in the Cuz1 AN1 ZnF.

<p>A) Sequence alignment of full-length Cuz1 with three human AN1 proteins: AIRAP (Zfand2A), AIRAP-L (Zfand2B), and Zfand1. Zinc chelating residues are highlighted in yellow. The LDFLP motif is highlighted in cyan. Asterisks, identical residues; double dots, highly similar residues; single dots, similar residues. B) Position of the LDFLP motif within the AN1 ZnF. The left panel shows the largely surface exposed LDFLP residues with their side chains. The right panel indicates hydrophobicity in green. Note the hydrophobic patch (in dark green) associated with the LDFLP motif, whereas the remainder of the structure shows a largely unremarkable hydrophobicity distribution.</p

Expression and Purification of the Cuz1 AN1 Zinc Finger Domain.

<p>A) Schematic diagram of the Cuz1 protein. UBL, ubiquitin-like domain. B) Purified Cuz1 AN1 ZnF protein (15 μg) as analyzed by SDS-PAGE followed by Coomassie staining. C) Size exclusion chromatography of purified Cuz1 AN1 ZnF protein. Molecular weight size standards are shown for reference. D) ¹⁵N-HSQC spectrum of the ¹⁵N-labeled Cuz1 AN1 ZnF domain sample showing assignments of Cuz1 residue resonance peaks. Backbone amide and sidechain peaks are labeled in red and green, respectively, and peaks from the cloning tag are marked with a “x” sign. An expanded view of the central spectral region is shown at the lower right corner.</p

The LDFLP Motif Appears Dispensable for Cdc48- and Proteasome-Binding.

<p>A) Binding of GST-tagged Cuz1 or the Cuz1^LDFL→AAAA mutant to 12xHis-Sumo-tagged Cdc48 or to beads alone. Binding reactions were visualized by SDS-PAGE followed by immunoblot with anti-Cuz1 antibody (upper panel). Cdc48 levels were analyzed by SDS-PAGE followed by Coomassie staining (lower panel). Input levels of GST-Cuz1 species were visualized to ensure that equivalent amounts of protein were added to the binding assay, and to confirm the assignment of the bands attributed to bound GST-Cuz1. B) Binding of purified proteasome to GST-tagged Cuz1 or the Cuz1^LDFL→AAAA mutant, as visualized by SDS-PAGE followed by immunoblot. Upper panel, anti-proteasome subunit Rpn8. Lower panel, anti-Cuz1 immunoblot. Metal chelated GST-Cuz1 serves as a negative control. C) Superimposed ¹⁵N-HSQC spectra of the AN1 ZnF domains from wild-type Cuz1 (blue) and Cuz1^LDFL→AAAA mutant (red). The resonance peaks from mutated residues L24, D25, and L27 are clearly missing (F26 overlaps with another peak).</p

Solution Structure of the AN1 ZnF Showing a Compact Fold.

<p>A) Ribbon diagram of the Cuz1 AN1 ZnF domain. Beta strands 1–4 and helices 1–3 are indicated, as well as the two zinc atoms as green spheres. B) Top view (rotated 90° about the X-axis) showing the compactness of the Cuz1 AN1 ZnF domain. Note that the scales are the same for panels A and B. C) Sequence alignment of the Cuz1 AN1 ZnF domain showing secondary structure: blue arrows, beta strands; red cylinders, short alpha helices. Residues coordinating the first zinc cluster are colored in red, and those coordinating the second zinc cluster in green. D) Central position of Phe38 at the core of AN1 ZnF domain between the two zinc chelating clusters. The dotted blue line indicates a hydrogen bond between His48 and His42 which further bridges the two zinc clusters. E) Additional polar and hydrophobic interactions that contribute to the stability of the compact Cuz1 AN1 ZnF domain.</p

NMR statistics of 15 solution structures of Cuz1 AN1 ZnF^*.

<p>NMR statistics of 15 solution structures of Cuz1 AN1 ZnF^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163660#t001fn001" target="_blank">*</a>}.</p

Rigidity of the AN1-ZnF Domain.

<p>A) ¹⁵N-HMQC spectrum of histidine residue side-chain correlations showing the different tautomeric state patterns of zinc-coordinating His42 (blue) and His48 (red). Note the appearance of the His48 Hδ1 resonance peak that is protected from rapid exchanging with H₂O by a side-chain-to-main-chain hydrogen bond. B) Atomic model of the zinc-coordinating histidine tautomeric states. The black dotted line indicates the side-chain-to-main-chain hydrogen bond involving His42 and His48. C) 2D-NOESY spectra of the Cuz1 ZnF showing chemical exchange cross-peaks between Hδ1/Hδ2 and Hε1/Hε2 spins of Phe38 due to the slow ring-flipping rate, marked by the red dotted lines. Note that the peaks are more broadened at 45°C than at 25°C due to faster chemical exchange, consistent with increasing ring-flipping rates.</p

Discovery and Characterization of a Disulfide-Locked <i>C</i>₂‑Symmetric Defensin Peptide

We report the discovery of HD5-CD, an unprecedented <i>C</i>₂-symmetric β-barrel-like covalent dimer of the cysteine-rich host-defense peptide human defensin 5 (HD5). Dimerization results from intermonomer disulfide exchange between the canonical α-defensin Cys^II–Cys^IV (Cys⁵–Cys²⁰) bonds located at the hydrophobic interface. This disulfide-locked dimeric assembly provides a new element of structural diversity for cysteine-rich peptides as well as increased protease resistance, broad-spectrum antimicrobial activity, and enhanced potency against the opportunistic human pathogen Acinetobacter baumannii

NMR Solution Structure and Condition-Dependent Oligomerization of the Antimicrobial Peptide Human Defensin 5

Human defensin 5 (HD5) is a 32-residue host-defense peptide expressed in the gastrointestinal, reproductive, and urinary tracts that has antimicrobial activity. It exhibits six cysteine residues that are regiospecifically oxidized to form three disulfide bonds (Cys³–Cys³¹, Cys⁵–Cys²⁰, and Cys¹⁰–Cys³⁰) in the oxidized form (HD5_ox). To probe the solution structure and oligomerization properties of HD5_ox, and select mutant peptides lacking one or more disulfide bonds, NMR solution studies and analytical ultracentrifugation experiments are reported in addition to <i>in vitro</i> peptide stability assays. The NMR solution structure of HD5_ox, solved at pH 4.0 in 90:10 H₂O/D₂O, is presented (PDB: 2LXZ). Relaxation <i>T</i>₁/<i>T</i>₂ measurements and the rotational correlation time (τ_c) estimated from a ¹⁵N-TRACT experiment demonstrate that HD5_ox is dimeric under these experimental conditions. Exchange broadening of the Hα signals in the NMR spectra suggests that residues 19–21 (Val¹⁹–Cys²⁰–Glu²¹) contribute to the dimer interface in solution. Exchange broadening is also observed for residues 7–14 comprising the loop. Sedimentation velocity and equilibrium studies conducted in buffered aqueous solution reveal that the oligomerization state of HD5_ox is pH-dependent. Sedimentation coefficients of ca. 1.8 S and a molecular weight of 14 363 Da were determined for HD5_ox at pH 7.0, supporting a tetrameric form ([HD5_ox] ≥ 30 μM). At pH 2.0, a sedimentation coefficient of ca. 1.0 S and a molecular weight of 7079 Da, corresponding to a HD5_ox dimer, were obtained. Millimolar concentrations of NaCl, CaCl₂, and MgCl₂ have a negligible effect on the HD5_ox sedimentation coefficients in buffered aqueous solution at neutral pH. Removal of a single disulfide bond results in a loss of peptide fold and quaternary structure. These biophysical investigations highlight the dynamic and environmentally sensitive behavior of HD5_ox in solution, and provide important insights into HD5_ox structure/activity relationships and the requirements for antimicrobial action