Conformation of the sebacyl β¹Lys82-β²Lys82 crosslink in T-state human hemoglobin

The crystal structure of human T state hemoglobin crosslinked with bis(3,5-dibromo-salicyl) sebacate has been determined at 1.9 Å resolution. The final crystallographic R factor is 0.168 with root-mean-square deviations (RMSD) from ideal bond distance of 0.018 Å. The 10-carbon sebacyl residue found in the β cleft covalently links the two βLys82 residues. The sebacyl residue assumes a zigzag conformation with cis amide bonds formed by the NZ atoms of βLys82's and the sebacyl carbonyl oxygens. The atoms of the crosslink have an occupancy factor of 1.0 with an average temperature factor for all atoms of 34 β². An RMSD of 0.27 for all CA's of the tetramer is observed when the crosslinked deoxyhemoglobin is compared with deoxyhemoglobin refined by using a similar protocol, 2HHD [Fronticelli et al. J. Biol. Chem. 269: 23965-23969, 1994]. Thus, no significant perturbations in the tertiary or quaternary structure are introduced by the presence of the sebacyl residue. However, the sebacyl residue does displace seven water molecules in the β cleft and the conformations of the β¹Lys82 and β²Lys82 are altered because of the crosslinking. The carbonyl oxygen that is part of the amide bond formed with the NZ of β²Lys82 forms a hydrogen bond with side chain of β2Asn139 that is in turn hydrogen-bonded to the side chain of β²Arg104. A comparison of the observed conformation with that modeled [Bucci et al. Biochemistry 35:3418-3425, 1996] shows significant differences. The differences in the structures can be rationalized in terms of compensating changes in the estimated free-energy balance, based on differences in exposed surface areas and the observed shift in the side- chain hydrogen-bonding pattern involving β²Arg104, β²Asn139, and the associated sebacyl carbonyl group.