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Background: Nitrophorins are nitric oxide (NO) transport proteins from the saliva of blood-feeding insects, which act as vasodilators and anti-platelet agents. Rhodnius prolixus, an insect that carries the trypanosome that causes Chagas' disease, releases four NO-loaded nitrophorins during blood feeding, whereupon the ligand is released into the bloodstream or surrounding tissue of the host. Histamine, a signaling molecule released by the host upon tissue damage, is tightly bound by the nitrophorins; this may facilitate the release of NO and reduce inflammation in the host., Results: Recombinant nitrophorin 4 (NP4) was expressed in Escherichia coli, reconstituted with heme, and found to bind NO and histamine in a manner similar to that of the natural protein. The crystal structure of NP4 revealed a lipocalin-like eight-stranded beta barrel, with heme inserted into one end of the barrel. His59 ligates the proximal site on the heme, a solvent molecule (NH3) ligates the distal site, and three additional solvent molecules occupy the distal pocket. Buried in the protein interior are Glu55 and three solvent molecules. A detailed comparison with other lipocalins suggests that NP4 is closely related to the biliverdin-binding proteins from insects., Conclusions: The nitrophorins have a unique hemoprotein structure and are completely unlike the globins, the only other hemoproteins designed to transport dissolved gases. Compared with the recently described structure of NP1, the NP4 structure is considerably higher resolution, confirms the unusual placement of ionizable groups in the protein interior, and clarifies the solvent arrangement in the distal pocket. It also provides a striking example of structural homology where sequence homology is minimal.

Background: Human thioredoxin reduces the disulfide bonds of numerous proteins in vitro, and can activate transcription factors such as NFkB in vivo. Thioredoxin can also act as a growth factor, and is overexpressed and secreted in certain tumor cells., Results: Crystal structures were determined for reduced and oxidized wild type human thioredoxin (at 1.7 and 2.1 A nominal resolution, respectively), and for reduced mutant proteins Cys73-->Ser and Cys32-->Ser/Cys35-->Ser (at 1.65 and 1.8 A, respectively). Surprisingly, thioredoxin is dimeric in all four structures; the dimer is linked through a disulfide bond between Cys73 of each monomer, except in Cys73-->Ser where a hydrogen bond occurs. The thioredoxin active site is blocked by dimer formation. Conformational changes in the active site and dimer interface accompany oxidation of the active-site cysteines, Cys32 and Cys35., Conclusions: It has been suggested that a reduced pKa in the first cysteine (Cys32 in human thioredoxin) of the active-site sequence is important for modulation of the redox potential in thioredoxin. A hydrogen bond between the sulfhydryls of Cys32 and Cys35 may reduce the pKa of Cys32 and this pKa depression probably results in increased nucleophilicity of the Cys32 thiolate group. This nucleophilicity, in tum, is thought to be necessary for the role of thioredoxin in disulfide-bond reduction. The physiological role, if any, of thioredoxin dimer formation remains unknown. It is possible that dimerization may provide a mechanism for regulation of the protein, or a means of sensing oxidative stress.