Structural basis for the NAD-hydrolysis mechanism and the
ARTT-loop plasticity of C3 exoenzymes
Julie Ménétrey, Gilles Flatau, Patrice Boquet, André Ménez, and Enrico A. Stura
Electronic Supplementary Discussion
Structural rearrangements reassessment in C3 exoenzyme.
Three structural rearrangements have been reported for the C3 exoenzyme induced by NAD
binding: an overall flexure and two local conformational changes involving the PN loop and
the ARTT loop, respectively (Menetrey et al. 2002). The overall flexure was first described
for the actin-ADP-ribosyltransferase toxin, VIP2 and the C3 exoenzyme from C. botulinum
(C3bot1) upon NAD binding (Han et al. 1999; Menetrey et al. 2002). Similarly, such a flexure
has been observed for the C3 exoenzyme from S. aureus (C3stau2) (Evans et al. 2003). Note
that this movement can be easily observed when structures are superimposed on one of the
perpendicular -sheet (Fig. 1). Evans et al. have recently measured the magnitude of this
overall flexure for C3bot1 using alignment of rigid domains with HINGEFIND (Wriggers and
Schulten 1997), and have reported that it is considerably smaller than that suggested
previously (Evans et al. 2004). Such a difference is probably inherent to the superposition
method applied. Moreover, the magnitude of this movement is measured within the crystal
lattice and it is difficult to extrapolate to the situation in solution. In absence of lattice
restraints the magnitude could be more important. Such clasp and release movement is
supposed to allow residues from the active site to come closer to the nucleotide and thus favor
catalysis (Han et al. 1999; Ménétrey et al. 2002; Evans et al. 2003), and later allow the
hydrolysis products to leave and liberate the ADP-ribosylated target protein (Ménétrey et al.
2002). On this basis, we would expect this movement to be affected by NAD, its hydrolysis
products, and the target protein, differently before and after ADP-ribosylation. Thus, it is not
surprising to find that the C3 flexure is influenced by protein-protein contacts in the crystal,
which could mimick the movements that could take place upon Rho binding and detachment.
Our new results show that the presence of NAD is not associated with a defined flexure, but is
compatible with preservation of alternating clasp and release conformations.
References
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