Abstract
The Potato II (Pot II) family of proteinase inhibitors plays important roles in the constitutive and inducible defense of plants against predation by a wide range of pests. The structural basis of inhibition by a multidomain Pot II family inhibitor was revealed recently by the structure of the ternary complex between the two-headed tomato inhibitor-II (TI-II) and two molecules of subtilisin Carlsberg. Here we report the 2.15-A resolution crystal structure of the unbound form of TI-II that reveals significant conformational flexibility in the absence of bound proteinase molecules. The four independent copies of unbound TI-II in the asymmetric unit of the unit cell display a range of different conformations when compared with the bound form of the inhibitor, most strikingly in the orientations of the inhibitory domains and in the conformations of the reactive site loops. One of the two linker segments (residues 74 to 79) between the two domains as well as the adjacent beta-strand in Domain I (residues 80-85) is well ordered in all four copies of the unbound inhibitor, even though this region appeared to be disordered in the structure of the ternary complex. Conformational flexibility seen in the reactive site loops of unbound TI-II suggests a mechanism by which the inhibitor can balance the need for tight binding with the need for broad inhibitory function.
Highlights
The atomic coordinates and structure factors have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ
Conformational flexibility seen in the reactive site loops of unbound tomato inhibitor-II (TI-II) suggests a mechanism by which the inhibitor can balance the need for tight binding with the need for broad inhibitory function
Tomato inhibitor-II (TI-II)1 is a member of the Potato II (Pot II) proteinase inhibitor family of serine proteinase inhibitors (PIs)
Summary
Purification and Crystallization—TI-II was prepared from transgenic tomato plants that overexpressed a prosystemin transgene, resulting in the synthesis and accumulation of high levels of TI-II in the leaves (ϳ1 mg/ml leaf juice). The solvent content of the unbound inhibitor crystals was calculated to be 59, 45, or 32% if three, four, or five copies of TI-II were present in the asymmetric unit, respectively. Each rotation function solution was used to calculate a translation function using data to a maximum resolution of 3.5 Å, each yielding one clear solution (log-likelihood-gain scores of 22.5 and 14.9). There was clear electron density for the interdomain residues 74 – 85 in all four copies of TI-II in the asymmetric unit. Because these residues were not modeled in the structure of TI-II in complex with subtilisin, they were placed by manual inspection using the molecular graphics program XFIT [41]. Figures were prepared with MOLSCRIPT [45], BOBSCRIPT [46], and RASTER3D
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