Efficient enzyme catalysis depends on exquisite details of structure beyond those resolvable in typical medium- and high-resolution crystallographic analyses. Here we report synchrotron-based cryocrystallographic studies of natural substrate complexes of the flavoenzyme human glutathione, reductase (GR) at nominal resolutions between 1.1 and 0.95 angstrom that reveal new aspects of its mechanism. Compression in the active site causes overlapping van der Waals radii and distortion in the nicotinamide ring of the NADPH substrate, which enhances catalysis via stereoelectronic effects. The bound NADPH and redox-active disulfide are positioned optimally on opposite sides of the flavin for a 1,2-addition across a flavin double bond. The new structures extend earlier observations to reveal that the redox-active disulfide loop in GR is an extreme case of sequential peptide bonds systematically deviating from planarity - a net deviation of 53 degrees across five residues. But this apparent strain is not a factor in catalysis, as it is present in both oxidized and reduced structures. Intriguingly, the flavin bond lengths in oxidized GR are intermediate between those expected for oxidized and reduced flavin, but we present evidence that this may not be clue to the protein environment but instead due to partial synchrotron reduction of the flavin by the synchrotron beam. Finally of more general relevance, we present evidence that the structures of synchrotron-reduced disulfide bonds cannot generally be used as reliable models for naturally reduced disulfide bonds. (C) 2008 Elsevier Ltd. All rights reserved.

Background: Glycerol kinase (GK) from Escherichia coli is a velocity-modulated (V system) enzyme that has three allosteric effecters with independent mechanisms: fructose-1,6-bisphosphate (FBP); the phosphocarrier protein IIA(Glc); and adenosine nucleotides. The enzyme exists in solution as functional dimers that associate reversibly to form tetramers, GK is a member of a superfamily of ATPases that share a common ATPase domain and are thought to undergo a large conformational change as an intrinsic step in their catalytic cycle, Members of this family include actin, hexokinase and the heat shock protein hsc70.Results: We report here the crystal structures of GK and a mutant of GK (Ala65-->Thr) in complex with glycerol and ADP, Crystals of both enzymes contain the same 222 symmetric tetramer. The functional dimer is identical to that described previously for the IIA(Glc)-GK complex structure, The tetramer interface is significantly different, however, with a relative 22.3 degrees rotation and 6.34 Angstrom translation of one functional dimer. The overall monomer structure is unchanged except for two regions: the IIA(Glc)-binding site undergoes a structural rearrangement and residues 230-236 become ordered and bind orthophosphate at the tetramer interface. We also report the structure of a second mutant of GK (Ile474-->Asp) in complex with IIA(Glc); this complex crystallized isomorphously to the wild type IIA(Glc)-GK complex. Site-directed mutants of GK with substitutions at the IIA(Glc)-binding site show significantly altered kinetic and regulatory properties, suggesting that the conformation of the binding site is linked to the regulation of activity.Conclusions: We conclude that the new tetramer structure presented here is an inactive form of the physiologically relevant tetramer. The structure and location of the orthophosphate-binding site is consistent with it being part of the FBP-binding site, Mutational analysis and the structure of the IIA(Glc)-GK(Ile474-->Asp) complex suggest the conformational transition of the IIA(Glc)-binding site to be an essential aspect of IIA(Glc) regulation.

Two features of the functional properties of lactoferrin are its ability to bind iron exceptionally tightly and the coupling of rigid-body domain movements to iron binding and release. The latter cause transitions between open and closed forms of the protein. Using site-directed mutagenesis and X-ray crystallography we have examined the importance of selected residues, including the iron ligands Asp 60 and His 253, the anion-binding Arg 121, and Pro 251 in the hinge region. Five mutants, D60S, R121S, R121E, H253M, and P251A, have been prepared in the context of the N-terminal half-molecule of human lactoferrin, LfN, and three-dimensional structures have been determined in each case. In D60S the mutation leads to weakened iron binding because a water molecule binds to the iron atom in place of Asp 60. Interdomain interactions are also weakened, and the loss of the Asp side-chain causes a significant change in domain closure; the domains move closer together by 7 degrees in the mutant. The R121S and R121E mutants show altered anion binding and very small changes in domain orientations. The H253M and P251A mutants show identical domain closure to wild-type LfN, but the iron site is altered in Hi253M; the Met 253 side-chain is not bound to iron, leaving a 5-coordinate site. These results are interpreted in terms of the roles of each of the residues in iron binding and release.

Lactoferrin is a protein that binds iron with great affinity, yet is also able to release it. It also binds a variety of other metal ions and anions. In order to investigate its mechanisms of binding and release, and the reasons for its versatility in binding, we have undertaken X-ray crystallographic studies on various forms of lactoferrin. The structure of a new crystal form of apolactoferrin, at 3.5-Angstrom resolution, has shown that in each lobe the binding cleft is in an open state, but that the size of the conformational change, compared with diferric lactoferrin, varies: a domain rotation of 54 degrees in the N-lobe and 18 degrees in the C-lobe. Comparison with the previously determined apolactoferrin structure, in which the C-lobe is closed, leads to a dynamic model for iron binding. The crystal structure of oxalate-substituted diferric lactoferrin shows that larger anions can be accommodated without affecting domain closure, although the two binding sites adjust differently. Solution studies also indicate that larger cations, such as Ce4+, may also be able to bind within the same closed structure. In this case, Ce3+ is oxidized to Ce4+ when it binds to lactoferrin, with a visible spectrum similar to those of Fe3+, Mn3+, and Co3+. Crystallographic binding studies using ruthenium complexes with antitumor activity show that these bind with high affinity in the binding cleft of apolactoferrin and more weakly in nonspecific external sites. This suggests possible uses of lactoferrin in drug delivery.

A conserved arginine residue helps to form the synergistic anion binding site in transferrins. To probe the importance of this residue for anion binding and iron binding, Arg 121 has been mutated to Ser and Glu in N-terminal half-molecule of human lactoferrin. The two mutants, R121S and R121E, have been expressed, purified, and crystallized. Their three-dimensional structures have been determined by X-ray diffraction at 2.3 and 2.5 A resolution, respectively. The structures were determined by molecular replacement and were refined by restrained least squares methods to final R values of 0.185 and 0.204. Both mutants still bind iron but with decreased stability. The crystal structures show that destabilization of iron binding probably results from disruption of the anion binding site; mutation of Arg 121 removes one wall of the anion binding pocket and causes the synergistic carbonate ion to be displaced 0.5 A from its position in the wild-type protein. In the process it becomes partially detached from the helix N-terminus that forms the rest of the anion binding site.

The three-dimensional structures of two cytochromes c' have been determined in order to analyse the common features of proteins of this family and their relationship with other four-helix bundle structures. The structure of cytochrome c' from Alcaligenes sp was determined by molecular replacement supplemented with the iron anomalous scattering and the use of a single isomorphous heavy-atom derivative, and was refined using synchrotron data to 1.8 A resolution. The final model, comprising 956 protein atoms (one monomer) and 89 water molecules, has a final R value of 0.188 for all data in the range 20.0-1.8 A resolution (14 673 reflections). The structure of the cytochrome c' from Alcaligenes denitrificans is isomorphous and essentially identical (r.m.s. deviation for all atoms 0.36 A). Although its amino-acid sequence has not been determined chemically, only four differences from that of Alcaligenes sp cytochrome c' were identified by the X-ray analysis. The final model for Alcaligenes denitrificans cytochrome c', comprising 953 protein atoms and 75 water molecules, gave a final R factor of 0.167 for all data in the range 20.0-2.15 A (8220 reflections). The cytochrome c' monomer forms a classic four-helix bundle, determined by the packing of hydrophobic side chains around the enclosed haem group. There are very few cross-linking hydrogen bonds between the helices, the principal side-chain hydrogen bonding involving one of the haem propionates and a conserved Arg residue. The cytochrome c' dimer is created by a crystallographic twofold axis. Monomer-monomer contacts primarily involve the two A helices, with size complementarity of side chains in a central solvent-excluded portion of the interface and hydrogen bonding at the periphery. Both species have a pyroglutamic acid N-terminal residue. The haem iron is five-coordinate, 0.32 A out of the haem plane towards the fifth ligand, His120. The unusual magnetic properties of the Fe atom may be linked to a conserved basic residue, Arg124, adjacent to His120.

The crystal structure of a site-specific mutant of the N-terminal half-molecule of human lactoferrin, LfN, in which the iron ligand Asp60 has been mutated to Ser, has been determined at 2.05 Å resolution in order to determine the effects of the mutation on iron binding and domain closure. Yellow monoclinic crystals of the D60S mutant, in its iron-bound form, were prepared, and have unit cell dimensionsa=110.2 Å,b=57.0 Å,c=55.2 Å, β=97.6°, space groupC2, with one molecule of 333 residues in the asymmetric unit. The structure was determined by molecular replacement, using the wild-type LfNas search model, and was refined by restrained least-squares methods. The final model, comprising 2451 protein atoms (from residues 2 to 315) one Fe3 +and one CO32−, and 107 water molecules, gives anR-factor of 0.175 for all data in the resolution range 20.0 to 2.05 Å. The model conforms well with standard geometry, having root-mean- square deviations of 0.014 Å and 1.2° from standard bond lengths and angles. The structure of the D60S mutant deviates in two important respects from the parent LfNmolecule. At the mutation site the Ser side-chain neither binds to the iron atom nor makes any interdomain contact as the substituted Asp does; instead a water molecule fills the iron coordination site and participates in interdomain hydrogen bonding. The domain closure is also changed, with the D60S mutant having a more closed conformation. Consideration of crystal packing suggests that the altered domain closure is a genuine molecular property but both the iron coordination and interdomain contacts are consistent with weakened iron binding in the mutant. The implications for iron binding in transferrins generally are discussed.

Background: Haemopexin is a serum glycoprotein that binds haem reversibly and delivers it to the liver where it is taken up by receptor-mediated endocytosis. Haemopexin has two homologous domains, each having a characteristic fourfold internal sequence repeat. Haemopexin-type domains are also found in other proteins, including the serum adhesion protein vitronectin and various collagenases, in which they mediate protein-protein interactions.Results: We have determined the crystal structure of the C-terminal domain of haemopexin at 1.8 Angstrom resolution. The domain is folded into four beta-leaflet modules, arranged in succession around a central pseudo-fourfold axis. A funnel-shaped tunnel through the centre of this disc-shaped domain serves as an ion-binding site.Conclusions: A model for haem binding by haemopexin is proposed, utilizing an anion-binding site at the wider end of the central tunnel, together with an associated cleft. This parallels the active-site location in other beta-propeller structures. The capacity to bind both cations and anions, together with the disc shape of the domain, suggests that such domains may be used widely for macromolecular recognition.