Mapping protein-protein interactions by localized oxidation: consequences of the reach of hydroxyl radical

Abstract

Hydroxyl radicals generated from a variety of methods, including not only synchrotron radiation but also Fenton reactions involving chelated iron, have become an accepted macromolecular footprinting tool. Hydroxyl radicals react with proteins via multiple mechanisms that lead to both polypeptide backbone cleavage events and side chain modifications (e.g., hydroxylation and carbonyl formation). The use of site-specifically tethered iron chelates can reveal protein-protein interactions, but the interpretation of such experiments will be strengthened by improving our understanding of how hydroxyl radicals produced at a point on a protein react with other protein sites. We have developed methods for monitoring carbonyl formation on proteins as a function of distance from a hydroxyl generator, iron-(S)-1-[p-(bromoacetamido)benzyl]EDTA (FeBABE), conjugated to an engineered cysteine residue. After activation of the chelated iron with ascorbate and peroxide produces new protein carbonyl groups, their positions can be identified using element-coded affinity tagging (ECAT), with carbonyl-specific tags {e.g., rare earth chelates of (S)-2-[4-(2-aminooxy)acetamidobenzyl]-1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (AOD)} that allow for affinity purification, identification, and relative quantitation of oxidation sites using mass spectrometry. Intraprotein oxidation of single-cysteine mutants of Escherichia coli sigma(70) by tethered FeBABE was used to calibrate the reach of hydroxyl radical by comparison to the crystal structure; the application to protein-protein interactions was demonstrated using the same sigma(70) FeBABE conjugates in complexes with the RNA polymerase core enzyme. The results provide fundamental information for interpreting protein footprinting experiments in other systems.

Scheme 1

FeBABE attached to a Cys side chain on a protein R. X is an activated oxygen ligand, which may produce a hydroxyl radical or other reactive species, even including a nucleophile (5). Rotation relative to the polypeptide backbone is possible around several single bonds. When the FeBABE is extended, the distance from the Cys sulfur to Fe is approximately 12Å.

Scheme 2

Carbonyl affinity tag MAOD, where M is a monoisotopic rare earth, showing how it is used to tag carbonyl groups produced by hydroxyl radical chemistry and identify their location in the protein sequence (13, 21).

Figure 1

Example of O-ECAT quantitation. During this experiment the oxidized protein was tagged with equimolar HoAOD and PrAOD so the theoretical HoAOD/PrAOD ratio was 1. First, the identity of the tagged peptide was manually confirmed after initially using Sequest (e.g. see panel A for the identification of the HoAOD tagged peptide). Next, the ion chromatogram from the narrow region of the monoisotopic peak of each parent was extracted (B and C). The intensities of the integrated ion signals were used to quantify O-ECAT peptide pairs. In this example, the HoAOD peptide has a signal intensity of 1.43E4 (B), while the PrAOD peptide has a signal of 1.53E4 (C), so the ratio of the intensities is 0.93.

Figure 2

The relative number of different potential reaction events, $W\left(r\right)=4\pi r^2{\stackrel{‒}{P}}_{rx}\left(r\right)$, among solute molecules that may be encountered by hydroxyl radicals as a function of distance (Å) from a point source, in 10% v/v glycerol (solid curve at lower left corner), 1% v/v glycerol (dashed curve), and 0.1% v/v glycerol (dot-dashed curve). The vertical scale is arbitrary. Glycerol is considered part of the solvent. Also shown, for a 5 μM protein solution, are vertical lines marking the average protein-protein nearest-neighbor distance nnd = 384Å, and distances one standard deviation (139Å) closer, nnd – σ, and two standard deviations closer, nnd – 2σ. For a 5 μM solution containing 10% v/v glycerol, hydroxyl radical encounters are effectively confined to the protein complex from which the radical originates.

Figure 3

Oxidized and tagged amino-acid residues mapped onto the crystal structure of a major fragment of σ⁷⁰ (PDB file: 1SIG) for three single-Cys mutants (132C, 376C, 422C). On the left side, the red sphere in each image represents the α-carbon of the single cysteine residue. The α-carbons of oxidized and tagged side chains are shown as green spheres, while the α-carbons of the oxidized and tagged N terminal residues resulting from backbone cleavage are shown as orange spheres. The identity of each amino acid residue is displayed, followed by the distance in Å from the α-carbon of the Cys residue to the α-carbon of the oxidized and tagged amino acid residue. Backbone cleavage determined from western blots (25) is shown as yellow ribbon (±3 residue std dev). On the right side, the space-fill images include information regarding the ratio of intensities of oxidation produced by FeBABE to that produced by FeEDTA -- e.g., 7.1× indicates that the FeBABE cleavage produced a 7.1× higher yield of that peptide -- for those residues where data are available. The sites efficiently oxidized by tethered FeBABE are colored blue, while the sites obscured from FeBABE but readily accessible to FeEDTA in solution are rendered in purple. If the ratio was not determined, colors match the images on the left side. The figures are rotated so that all the residues of interest can be seen in the space-fill model.

Figure 4

Oxidized and tagged residues mapped onto the crystal structure of to Thermus Aquaticus RNA polymerase holoenzyme (PDB file: 1L9U). The β subunit is colored blue, the β′ subunit is colored pink, the α₂ and ω subunits are gray, and the σ⁷⁰ subunit is green. The single cysteine side chain is shown in red. Oxidized and tagged side chains are shown as gray spheres. Oxidized and tagged N termini are shown as orange spheres. Previously determined backbone cleavage data (strong cuts) are shown as 20-residue segments of small spheres (uncertainty due to assignment by electrophoretic mobility) (6).