S-Nitrosylation-induced Conformational Change in Blackfin Tuna Myoglobin

Abstract

S-Nitrosylation is a post-translational protein modification that can alter the function of a variety of proteins. Despite the growing wealth of information that this modification may have important functional consequences, little is known about the structure of the moiety or its effect on protein tertiary structure. Here we report high-resolution x-ray crystal structures of S-nitrosylated and unmodified blackfin tuna myoglobin, which demonstrate that in vitro S-nitrosylation of this protein at the surface-exposed Cys-10 directly causes a reversible conformational change by "wedging" apart a helix and loop. Furthermore, we have demonstrated in solution and in a single crystal that reduction of the S-nitrosylated myoglobin with dithionite results in NO cleavage from the sulfur of Cys-10 and rebinding to the reduced heme iron, showing the reversibility of both the modification and the conformational changes. Finally, we report the 0.95-Å structure of ferrous nitrosyl myoglobin, which provides an accurate structural view of the NO coordination geometry in the context of a globin heme pocket.Protein S-nitrosylation, or the formation of an S-NO bond involving the sulfur of a cysteine residue, is an important posttranslational modification. Numerous proteins whose functions are altered by S-nitrosylation have been identified, including enzymes, transcription factors, receptors, channels, and structural proteins (reviewed in Ref.

An Amber Force Field for S-Nitrosoethanethiol That Is Transferable to S-Nitrosocysteine

“…Structures of myoglobin (Mb) with 10 CysSNO residue were extracted from 2nrm.pdb. 4 Iron was removed from the heme and the pyrrol nitrogens were deprotonated. Atomic charges of the resulting demetallated heme were calculated according to a RESP protocol.…”

“…Interested in redox regulatory proteins, we previously reported MD simulations of S-nitrosated thioredoxin 9 in which the CC-SN dihedral lies in between 185 and 289 degrees. 3 Other S-nitrosated proteins, 4 however, may well have other values for the dihedral angles so that it is desirable to have a better force field that accounts for the whole range of dihedral angles. In this report we developed an Amber force field for EtSNO that can be transferred to CysSNO reproducing the CC-SN and CS-NO torsional energies of CysSNO in both α and β-conformations for the whole range of CC-SN and CS-NO dihedrals.…”

“…Unfortunately three dimensional structures have been solved only for a few S-nitrosated proteins. [2][3][4][5] Therefore molecular dynamics (MD) simulation with good force field parameters may serve as an alternative method for probing the structural alteration in S-nitrosated proteins.…”

“…In fact Cys10, the site of S-nitrosation, lies far from the heme so that the freezing itself is not expected to affect the conformations of 10 CysSNO. To account for the two conformations of 10 CysSNO, 4 we performed MD simulations of Mb with 10 CysSNO whose initial CC-SN dihedrals are -88 and 124 degrees. As shown in Fig.…”

“…10 We could not deduce any preferred conformations, however, because 3D structures are available only for a few S-nitrosated proteins. [2][3][4][5] We therefore adopted the values of ϕ, ψ, and χ1 (i.e. C-N-CA-CB dihedral) from the conformation of unmodified CysSH that is listed in the Amber-99 force field.…”