Year: 2019
Type: letter
Abstract: Wang1 doubts our2, 3 claim that the unexpected feature identified as a bridge between the side chains of Lys and Cys in the crystal structure of HPP and some other structures in the PDB is a methylene moiety. In his interpretation, these residues are connected by an O atom. Both claims, Wang's and ours, are supported by indirect reasoning, since even mass spectroscopy cannot decisively differentiate between these two possibilities, taking into account that the difference between the mass of the –CH2– and –O– moieties is about 2 with unknown protonation state of the protein. It must be emphasized that in our original study,2 mass spectrometry was used to confirm that the dimerization of HPP is covalent but not to identify its chemical nature. Our argument (concerning HPP, and overlooked by Wang) was that the appearance of a significant electron density peak between the Lys Nζ and Cys Sγ atoms was enormously accelerated if the crystals were subjected to vapors of formaldehyde which is a potent agent for cross-linking amines with thiols. In fact, our structure 5EQA discussed by Wang resulted from an overnight incubation of HPP crystals in formaldehyde vapors. It is likely that Wang is somewhat biased in his tendency to see the X-ray-induced addition of oxygen atoms to protein residues.4 In science, any bias can have dangerous consequences, and we all must avoid acting as Piglet, who was biased to see a Horrible Heffalump instead of his friend, Winnie-the-Pooh, who had his head stuck in a honey jar.5 In comment on my paper on the covalent Cys-O-Lys bridges,1 Ruszkowski and Dauter2 caution: (i) whether I have overlooked the fact that diffraction data being analyzed were collected from an histidinol phosphate phosphatase crystal incubated overnight with vapors of formaldehyde, (ii) whether mass spectrometric data they have obtained can decisively differentiate between the two possibilities of chemical identity of being either –CH2– or –O– at the X position of the Cys-X-Lys bridges, (iii) whether I have a biased view on X-ray radiation-induced addition of oxygen atoms to proteins that may have crowded my interpretation of their data, and (iv) whether my interpretation was based on indirect reasoning, as was their claim. I want to emphasize that my crystallographic analysis of their electron density maps on the total number of electrons distributed around the nuclear center of species X is a direct observation, not indirect reasoning. If species were –CH2–, the number of electrons around the C center would never exceed that of other reference C centers nearby. If the number of electrons around the X center exceeds that of other C centers nearby, which my analysis shows indeed to be the case, species X can only be an –O– atom. Furthermore, I discovered that Ruszkowski and Dauter made a mistake in interpreting the mass of the adductive that cross-link two HPP monomers by two units.3 The incorrect mass supports their original claim,3 but the correct mass supports my new interpretation.1 However, I also note that an alternative interpretation of the correct mass unit also exists. Given the fact that mass spectrometry measures mass unit according to the distribution of mass-to-charge ratios, resulting mass unit is always calibrated with zero-charge species, regardless the actual protonation state of species in solution. Although X-ray radiation-induced addition of oxygen atoms to the protein has been observed in this protein,1 I never suggest in my paper that this is responsible for the formation of Cys-O-Lys bridges. X-ray radiation-induced oxygen addition tends to target locations that are often resistant to spontaneous oxidation such as the Cβ position of Arg-194. Analysis of all other Cys side chains in the HPP protein shows that they all have been oxidized, suggesting that spontaneous oxidation, including formation of Cys-O-Lys bridges, may have occurred during sample preparation, but before X-ray irradiation. Indeed, it is well known that formaldehyde can cross-link primary amines. However, it remains to be established whether formaldehyde could modify already oxidized primary amines, or oxidized Cys side chains, or the already formed Cys-O-Lys bridges. Even though this may be possible under certain circumstance, the result of my analysis on the total number of electrons does not support it. The incubation of an HPP crystal overnight with vapors of formaldehyde does not guarantee the replacement of –CH2– insertion at the already-formed Cys-O-Lys bridges. Finally, the majority of protein structures containing Cys-X-Lys bridges they have identified4 did not include an incubation step with vapors of formaldehyde. My bond-energy analysis1 suggests that the formation of the Cys-O-Lys bridges is a spontaneous process whereas the formation of the Cys-CH2-Lys bridges is not. JIMIN WANG Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, 06520 1. Wang J (2018) Crystallographic identification of spontaneous oxidation intermediates and products of protein sulfhydryl groups. Protein Sci 28:472–477. 2. Ruszkowski M, Dauter Z (2019) Comment on Wang's paper on the covalent Cys-X-Lys bridges. Protein Sci 28. 3. Ruszkowski M, Dauter Z (2016) Structural studies of Medicago truncatula histidinol phosphate phosphatase from inositol monophosphatase superfamily reveal details of penultimate step of histidine biosynthesis in plants. J Biol Chem 291:9960–9973. 4. Ruszkowski M, Dauter Z (2016) On methylene-bridged cysteine and lysine residues in proteins. Protein Sci 25:1734–1736.
Source: Protein Science
DOI: 10.1002/pro.3576
Authors: Milosz Ruszkowski, Zbigniew Dauter
Institutions: Argonne National Laboratory, National Cancer Institute
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: bronze
OpenAlex ID: W2912157758