Abstract
Multidomain proteinase inhibitors play critical roles in the defense of plants against predation by a wide range of pests. Despite a wealth of structural information on proteinase-single domain inhibitor interactions, the structural basis of inhibition by multidomain proteinase inhibitors remains poorly understood. Here we report the 2.5-A resolution crystal structure of the two-headed tomato inhibitor-II (TI-II) in complex with two molecules of subtilisin Carlsberg; it reveals how a multidomain inhibitor from the Potato II family of proteinase inhibitors can bind to and simultaneously inhibit two enzyme molecules within a single ternary complex. The N terminus of TI-II initiates the folding of Domain I (Lys-1 to Cys-15 and Pro-84 to Met-123) and then completes Domain II (Ile-26 to Pro-74) before coming back to complete the rest of Domain I (Pro-84 to Met-123). The two domains of TI-II adopt a similar fold and are arranged in an extended configuration that presents two reactive site loops at the opposite ends of the inhibitor molecule. Each subtilisin molecule interacts with a reactive site loop of TI-II through the standard, canonical binding mode. Remarkably, a significant distortion of the active site of subtilisin is induced by the presence of phenylalanine in the P1 position of reactive site loop II of TI-II. The structure of the TI-II.(subtilisin)2 complex provides a molecular framework for understanding how multiple inhibitory domains in a single Potato II type proteinase inhibitor molecule from the Potato II family act to inhibit proteolytic enzymes.
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
The inhibitory properties toward serine proteinases of these proteinase inhibitors (PIs) have already been exploited with varying degrees of success for the production of transgenic plants overexpressing the PIs in an attempt to control pests (9 –13)
The release of jasmonic acid leads to the activation of several signaling pathways that in turn lead to the production of more jasmonic acid and H2O2 and the synthesis of PIs [26]
Summary
Purification and Crystallization—Tomato inhibitor-II was prepared from transgenic tomato plants that overexpressed a prosystemin transgene, which resulted in the synthesis and accumulation of high levels of TI-II in the leaves (ϳ1 mg/ml leaf juice). The structure of subtilisin Carlsberg (PDB code 1SCN), with all the side chains and temperature factors retained, was used as the search model for molecular replacement calculations. Each rotation function solution was used to calculate a translation function using data to a maximum resolution of 3 Å, yielding two solutions (R-factors of 0.485 and 0.503 after rigid-body refinement). The relative positions of the two subtilisin molecules in the asymmetric unit were determined using a phased translation function, yielding a single solution (R-factor of 0.396). Rotation and translation searches using a search model for either a single domain or both domains of the inhibitor, as constructed from PCI-I (PDB code 4SGB) [35], did not reveal any clear solutions.
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