Crystallization of an apo form of human arginase: using all the tools in the toolbox simultaneously

Abstract

Arginase (EC 3.5.3.1) is an aminohydrolase that acts on l-arginine to produce urea and ornithine. Two isotypes of the enzyme are found in humans. Type I is predominantly produced in the liver and is a homotrimer of 35 kDa subunits. Human arginase (hArginase) I is seen to be up-regulated in many diseases and is a potential therapeutic target for many diverse indications. Previous reports of crystallization and structure determination of hArginase have always included inhibitors of the enzyme: here, the first case of a true apo crystal form of the enzyme which is suitable for small-molecule soaking is reported. The crystals belonged to space group P2 1 2 1 2 1 and have approximate unit-cell parameters a = 53, b = 67.5, c = 250 Å . The crystals showed slightly anisotropic diffraction to beyond 2.0 Å resolution.

Microseed matrix screening for optimization in protein crystallization: what have we learned?

“…Our experiments demonstrated that the cross-linked seeds retained their capacity to induce crystal formation. This has also been confirmed by Newman et al (2011) in work on human arginase.…”

“…Since the introduction of MMS in 2004, a number of reports (Obmolova et al, 2010;Persson et al, 2010;Newman et al, 2011) have confirmed that it is a viable optimization alternative. It can produce high-quality crystals from poor starting points that are not amenable to the classical optimization strategies.…”

Combining Counter-Diffusion and Microseeding to Increase the Success Rate in Protein Crystallization

“…Although we selected only one of the crystal forms as seed stock, we found that both forms could be obtained from the same seeds (Figure 1A-a and C-c). Similar results have been obtained recently with human arginase, 18 but since we have not yet characterized those crystals by X-ray diffraction, we will not discuss this observation further. Crystal Growth & Design…”

Evaluating Protic Ionic Liquids as Protein Crystallization Additives

“…Using the PILs in conjunction with sparse matrix screens, such as the JCSG+ screen used in this study, increases the likelihood of identifying crystallization conditions which can be further optimized. This is a methodology which we are actively applying to the crystallization of proteins which are difficult to crystallize; these PILs have already been shown to be useful in our lab in generating crystals of diffraction quality for a difficult project …”