Photon Factory Activity Report 2005 #23 Part B (2006)
Crystallography

NW12A/2005G259

Crystal structure of a clip-domain serine protease, prophenoloxidase-activating
factor (PPAF)-IIShunfu Piao1, Young-Lan Song2, Jung Hyun Kim1, Sam Young Park3, Ji Won Park, Bok Leul Lee*1,
Byung-Ha Oh*2 and Nam-Chul Ha*1
1
Pusan National Univ., Jangjeon Dong, Geumjeong Gu, Busan, Korea
2
Pohang Univ. of Science and Technology, Pohang, Kyungbuk, Korea
3
Yokohama City Univ., Suehiro, Tsurumi-ku, Yokohama, Japan
Introduction
Serine protease (SP) cascades amplify signals fromphysiological or pathological responses in theextracellular region of vertebrates and invertebrates. SP
cascades drive various biological processes, especially inembryonic development and the innate immune responses
of invertebrates. SPs consist of a chymotrypsin-like SP
domain and one or two clip domains at the N-terminus.
Prophenoloxidase-activating factor (PPAF)-II, which
belongs to the noncatalytic clip-domain SP family, isindispensable for the generation of the active
phenoloxidase leading to melanization, a major defense
mechanism of insects. Here, the crystal structure ofPPAF-II reveals that the clip domain adopts a novel foldcontaining a central cleft, which is distinct from thestructures of defensins with a similar arrangement ofcysteine residues.
Experiments
Crystallization of PPAF-II was reported [1]. Data
collection and processed procedures were reported [2].
Results and Discussion
Overall structure of PPAF-II
The PPAF-II gene encodes a protein with 415 aminoacids. The N-terminal 24-amino-acid segment of theprotein functions as a signal peptide for secretion. The
mature protein (392 amino acids) is made up of two parts:
the clip domain including the flanking sequence
composed of 114 amino acids, and the SP-like (SPL)
domain composed of 278 amino acids. The final 2.0 Åresolution model contains residues 22–42 with an N-Figure1. Overall structure of PPAF-II. A ribbon representation of PPAFII structure. PPAF-II family-specific N-terminal segment is in yellow.
The four signature sequences are in magenta. The nonfunctional
catalytic triad residues (Gly-His-Asp) are shown in the ball-and-stick

glycosylated residue, 47–116, 129–173, and 180–415,
which include most of the clip and the SPL domains(Figure1). The missing residues constitute the loop
segments flanking the clip domain and a flexible loop in
the SPL domain. The small clip domain is bound to thelarger SPL domain via hydrogen bonds, hydrophobic
interactions, and a salt bridge. While the structure of theSPL domain of PPAF-II is similar to chymotrypsin-likeproteases that of the clip domain is a new fold according
to a database search using the program DALI.
Structure feature of clip domain

Figure 2. Comparison of the clip domain with human β-defensins 1. Leftand right are a schematic drawing of the clip domain and a schematic
drawing of β-defensins, respectively.

The clip domain is composed of a high portion of loops
and a central, four-stranded, irregular β-sheet. The
conserved three disulfide bonds knotting the loops andthe β-strands together appear critical for the structural
integrity of the central β-sheet that serves as the main
framework of the clip domain structure. It has beensuggested that clip domains may be structurally similar to
antimicrobial proteins, β-defensins, based on the identicalarrangement of the cysteine residues. We show here thatthe clip domain structure is distinctively different fromthose of β-defensins. First, β-defensins contain three,instead of four, β-strands forming the central antiparallel
β-sheet (Figure2). Second, while β2 and β3 are
antiparallel with each other in the structure of β-defensins,
they are parallel in the structure of the clip domain(Figure2).
References[1] S. Piao et al., Biochim Biophys Acta 1752: 103–106(2005).
[2] S. Piao et al., EMBO 24, 4404-4414(2005).
*bhoh@postech.ac.kr

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