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

6A, 18B, NW12/2003G112

Structure and Function of the ETD mutant by the X-ray Crystallography
Katsuo KATAYANAGI*, Yasunori KAMIYA, JingJie MAO, Yoshihiko HORI, Kenji YAMAMOTO,
Fuki HAGIWARA, Hiromi KITTAKA
Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University,Higashi-Hiroshima 739-8526, Japan

Introduction
Exfoliative toxin (ET) is one of the exotoxin, which
was secreted by staphylococcal species, and it causes
blisters in animal and human skin. Exfoliative toxins(ET’s), serotypes A (ETA) and B (ETB), from
Staphylococcal aureus cause staphylococcal scaled skin
syndrome (SSSS), or Ritter disease, and bullous impetigo
in neonates. Both types cause intraepidermal cleavage in
the granular layer, without epidarmal necrolysis orinflammatory response of skin. Recently, the mechanismof Exfoliative Toxin was found as act as serine protease.
The crystal structures of ETA and ETB have been
determined in 1999, and both types were shown tostructurally belong to the chymotrypsin family of serineproteases. Moreover, it has been shown that ETA digests
desmoglein I, which is one of the major desmosome
proteins present in the epidermal layer of human skin. On
the other hand, Motoyuki Sugai et al. found new serotype
ET in the process of analyzing S. aureus genes, anddesignated as Exfoliative Toxin D (ETD).
Results and Discussion
We have determined the three-dimensional structureof ETD, and clarified that its structure is closer to ETB
rather than ETA. In this study, we have crystallized theactive site mutant S189A, and we will elucidate thestructural background of these unknown functions of thenew ETD.
The crystal of ETD S189A was obtained as wild type.
But high resolution diffraction was obtained only fromlarge (more than 0.3mm diameter) crystal. Unfortunately,such large crystal was obtained only 1 batch among morethan 40 batches. Other ones were quite thin needle
crystals of only 0.01 mm diameter. Such needle crystals
diffracted less than 5 Å resolutions. Finally, 2.4 Åresolution data were obtained. This crystal belong to thespace group P61, and the lattice parameter wasa=b=122.10 Å, c=122.67 Å, and the Rsym was 0.102.
The molecular replacement method using AMoRe andMolRep were applied to determine the initial phase. By
comparing ETA and ETB, a quite unique loop (155-163),
which locates near the cataritic triad, was newly found in
the determined 3D structure of ETD. Thus, S189A (S189
is one of the catalytic triad) structure will show therelationship of serine protease activity and exfoliativeactivity.
Moreover, in order to clarify the structure-function
relationship, we made a crystal of loop modified ETDmutant. The loop was selected as discussed above (155163), and amino acid sequence of ETD loop wasmodified as that of ETB. The obtained crystal of this loop
mutant was quite thin less than 1 µm (area was 200x400µm), it diffracted x-ray over 2.7 Å resolution. This
crystal belong to the space group P212121, and the lattice
parameter was a=69.12 Å, b=104.01 Å, c=108.25 Å, andthe Rsym was 0.102. Structural analysis was performed
using molecular replacement method. According to theresult of the structural analysis, the loop structure of ETD
loop mutant became almost identical to the structure ofETB.
We have determined the new exfoliative toxin ETD ,its active site mutant, and ETB like loop modified mutant.
These structures are the start point to identify thestructural exfoliative activity from serin protease activity.
Based on these results, we are now expanding to makeET-dsg1 co-crystal.

* kkata@sci.hiroshima-u.ac.jp

Users' Report161