Molecular arrangements that accompany binding of rice xylanase inhibitor protein OsXIP and the Rhizopus oryzae GH11 xylanase RXyn2.

Plants have evolved xylanase inhibitor proteins as part of their defense mechanisms against phytopathogens. The rice xylanase inhibitor protein (OsXIP) is structurally similar to GH18 chitinase and homologous to wheat XIP-type inhibitor (XIP-I), which inhibits both GH10 and GH11 xylanases. Various inhibition and interaction analyses showed that OsXIP competitively inhibits the hydrolytic activity of GH11 xylanase RXyn2, but not the activity of GH10 xylanase RXyn1 from Rhizopus oryzae. The crystal structure of the OsXIP/RXyn2 complex showed that OsXIP, which has a (β/α)-barrel fold, extrudes the loop between α4 and β5 (Lα4β5) and inserts the loop into the xylotriose binding site (-3 to -1 subsite) formed by the inner β-sheet (palm) of RXyn2 jelly roll. The guanidyl group of Arg155 in Lα4β5 was shown to be critical for the inhibitory activity by mutational analysis. Notably, in the complex structure, the cylindrical cavity formed by the palm of RXyn2 jelly roll stacked upright on the loops at the N terminal ends of the β-strands of OsXIP (I-formation). On the other hand, in the complex structure of XIP-I and GH11 xylanase from Talaromyces funiculosus (XYNC), the cavity of XYNC laid tangentially to the part of the corresponding region of XIP-I through the Lα4β5 (T-formation). The dissociation constant of the OsXIP/RXyn2 complex was one-tenth of that of the XIP-I/XYNC complex (4.2 versus 41.5 nM). OsXIP may have adapted to bind and inhibit GH11 enzymes, which are resistant to the inhibition by XIP-I type proteins, by changing its binding mode.