Model bias in macromolecular crystal structures

Stellacyanins are blue (type I) copper glycoproteins that differ from other members of the cupredoxin family in their spectroscopic and electron transfer properties. Until now, stellacyanins have eluded structure determination. Here we report the three-dimensional crystal structure of the 109 amino acid, non-glycosylated copper binding domain of recombinant cucumber stellacyanin refined to 1.6 8, resolution. The crystallographic R-value for all 18,488 reflections ((T > 0) between 50-1.6 8, is 0.195. The overall fold is organized in two P-sheets, both with four P-strands. Two cy-helices are found in loop regions between P-strands. The P-sheets form a P-sandwich similar to those found in other cupredoxins, but some features differ from proteins such as plastocyanin and azurin in that the P-barrel is more flattened, there is an extra N-terminal a-helix, and the copper binding site is much more solvent accessible. The presence of a disulfide bond at the copper binding end of the protein confirms that cucumber stellacyanin has a phytocyanin-like fold. The ligands to copper are two histidines, one cysteine, and one glutamine, the latter replacing the methionine typically found in mononuclear blue copper proteins. The Cu-Gln bond is one of the shortest axial ligand bond distances observed to date in structurally characterized type I copper proteins. The characteristic spectroscopic properties and electron transfer reactivity of stellacyanin, which differ significantly from those of other well-characterized cupredoxins, can be explained by its more exposed copper site, its distinctive amino acid ligand composition, and its nearly tetrahedral ligand geometry. Surface features on the cucumber stellacyanin molecule that could be involved in interactions with putative redox partners are discussed.

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“….6 8,. Model atom positions were verified by the examination of conventional and simulated annealing omit maps (Hodel et al, 1992). …”

Section: Methodsmentioning

confidence: 99%

A missing link in cupredoxins: Crystal structure of cucumber stellacyanin at 1.6 Å resolution

et al. 1996

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“…The same procedure was used to locate five ureas in the dihydrofolate structure. At the end of refinement, simulated annealing omit maps were calculated for each denaturant (Hodel et al, 1992;Yennawar et al, 1994). Convincing density was observed for each denaturant in these maps.…”

Section: Crystalsmentioning

confidence: 99%

The effect of denaturants on protein structure

et al. 1997

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Virtually all studies of the protein-folding reaction add either heat, acid, or a chemical denaturant to an aqueous protein solution in order to perturb the protein structure. When chemical denaturants are used, very high concentrations are usually necessary to observe any change in protein structure. In a solution with such high denaturant concentrations, both the structure of the protein and the structure of the solvent around the protein can be altered. X-ray crystallography is the obvious experimental technique to probe both types of changes. In this paper, we report the crystal structures of dihydrofolate reductase with urea and of ribonuclease A with guanidinium chloride. These two classic denaturants have similar effects on the native structure of the protein. The most important change that occurs is a reduction in the overall thermal factor. These structures offer a molecular explanation for the reduction in mobility. Although the reduction is observed only with the native enzyme in the crystal, a similar decrease in mobility has also been observed in the unfolded state in solution (Makhatadze G, Privalov PL. 1992. Protein interactions with urea and guanidinium chloride: A calorimetric study. J Mol B i d 226491-505).Keywords: denaturants; dihydrofolate reductase; protein mobility; ribonuclease A Despite our routine use of them, our understanding of the molecular mechanism by which chemical denaturants like urea or guanidinium (Gua) cause a protein to unfold is still rather limited. As has been pointed out by others (Schellman, 1987), part of the problem is that it is difficult to measure binding constants when the protein concentration is M and the denaturant concentration is 4 M. High precision scanning calorimetry has provided one experimental solution to this problem (Makhatadze & Privalov, 1992) but relating the thermodynamic data produced in such an experiment to protein structure can be difficult (Alber, 1989). While it does seem clear that denaturants interact directly with the protein (Simpson & Kauzmann, 1953), it is also clear that at high concentrations these molecules can cause substantial changes to the behavior of the solvent itself (Breslow & Guo, 1990 teins by migrating into the interior of the protein and forming hydrogen bonds to atoms in the backbone (Hedwig et al., 1991). In support of such a model, the exchange rates of some peptide NH protons, which are found on both the surface and the interior of a protein, are decreased in the presence of urea (Kim & Woodward, 1993). In addition, crystallographic studies of diketopiperazine and urea show hydrogen bonding between the amide groups and the urea (Thayer et al., 1993). Other models have suggested that denaturants act by decreasing the hydrophobic effect (Wetlaufer et al., 1964;Roseman & Jencks, 1975), which, in this instance, is usually postulated to be the dominant force stabilizing protein structure. Finally, experiments by Kim and Woodward (1 993) clearly show that, in addition to interacting with peptide bonds, urea alters...

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“…Five percent of the data were set aside for cross-validation analysis (Briinger, 1992). After each refinement step, 2F0 -F, and F, -F, electron density maps were computed.…”

Section: Structure Determination and Refinementmentioning

confidence: 99%

Crystal structure of cutinase covalently inhibited by a triglyceride analogue

et al. 1997

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Cutinase from Fusarium solani is a lipolytic enzyme that hydrolyses triglycerides efficiently. All the inhibited forms of lipolytic enzymes described so far are based on the use of small organophosphate and organophosphonate inhibitors, which bear little resemblance to a natural triglyceride substrate. In this article we describe. the crystal structure of cutinase covalently inhibited by (R)-1,2-dibutyl-carbamoylglycero-3-O-p-nitrophenyIbutyI-phosphonate, a triglyceride analogue mimicking the first tetrahedral intermediate along the reaction pathway. The structure, which has been solved at 2.3 A, reveals that in both the protein molecules of the asymmetric unit the inhibitor is almost completely embedded in the active site crevice. The overall shape of the inhibitor is that of a fork: the two dibutyl-carbamoyl chains point towards the surface of the protein, whereas the butyl chain bound to the phosphorous atom is roughly perpendicular to the sn-1 and sn-2 chains. The sn-3 chain is accommodated in a rather small pocket at the bottom of the active site crevice, thus providing a structural explanation for the preference of cutinase for short acyl chain substrates.

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Model bias in macromolecular crystal structures

Cited by 398 publications

References 8 publications

A missing link in cupredoxins: Crystal structure of cucumber stellacyanin at 1.6 Å resolution

A missing link in cupredoxins: Crystal structure of cucumber stellacyanin at 1.6 Å resolution

The effect of denaturants on protein structure

Crystal structure of cutinase covalently inhibited by a triglyceride analogue

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