Structure-Function Analysis of Grass Clip Serine Protease Involved in Drosophila Toll Pathway Activation

Christine Kellenberger,Philippe Leone,Laurent Coquet,Thierry Jouenne,Jean-Marc Reichhart,Alain Roussel

doi:10.1074/jbc.m110.182741

Abstract

Grass is a clip domain serine protease (SP) involved in a proteolytic cascade triggering the Toll pathway activation of Drosophila during an immune response. Epistasic studies position it downstream of the apical protease ModSP and upstream of the terminal protease Spaetzle-processing enzyme. Here, we report the crystal structure of Grass zymogen. We found that Grass displays a rather deep active site cleft comparable with that of proteases of coagulation and complement cascades. A key distinctive feature is the presence of an additional loop (75-loop) in the proximity of the activation site localized on a protruding loop. All biochemical attempts to hydrolyze the activation site of Grass failed, strongly suggesting restricted access to this region. The 75-loop is thus proposed to constitute an original mechanism to prevent spontaneous activation. A comparison of Grass with clip serine proteases of known function involved in analogous proteolytic cascades allowed us to define two groups, according to the presence of the 75-loop and the conformation of the clip domain. One group (devoid of the 75-loop) contains penultimate proteases whereas the other contains terminal proteases. Using this classification, Grass appears to be a terminal protease. This result is evaluated according to the genetic data documenting Grass function.

Highlights

Some biological processes such as blood coagulation in mammals or development and immune responses in invertebrates occur after the amplification of a recognition signal by serine proteases (SP)2 that are organized in cascades [1, 2]
Structure of the Catalytic Domain of Grass—The structure of Grass consists of two domains, the clip and the catalytic domains connected by a linker comprising residues 91–118
The SP domain of Grass (Val119–Leu377) exhibits the characteristic polypeptide fold of trypsin-like SPs consisting of two ␤-barrels made of six ␤-strands stacked onto one another (Fig. 2A)

Summary

Crystal Structure of Toll Pathway Grass Clip Serine Protease

Two clip-SPs, namely Spaetzle-processing enzyme (SPE) and Grass, have been demonstrated to participate in this cascade [6, 7]. Proteases and PRRs have been isolated from T. molitor, and a signaling cascade triggering the Toll activation has been reconstituted in vitro It is composed of an apical modular SP named Tm-MSP and two clip-SPs, Tm-SAE and Tm-SPE. We recently undertook a systematic structural characterization of the extracellular components identified in the activation of the Drosophila Toll receptor [20, 21] In this context, we determined the crystal structure of the clip-SP Grass, in its zymogen form, which represents the first structure of a fulllength clip-SP. This enables us to predict the position, penultimate or terminal, of any clip-SP within a cascade Using this approach, we propose a new model for the role of Grass in Drosophila Toll pathway activation

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION