TORPEDO CALIFORNICA ACETYLCHOLINESTERASE IN COMPLEX WITH A NON HYDROLYSABLE SUBSTRATE ANALOGUE, 4-OXO-N,N,N-TRIMETHYLPENTANAMINIUM (OTMA) - ORTHORHOMBIC SPACE GROUP-DATASET A AT 100K (2fcj)
Although x-ray crystallography is the most widely used method for macromolecular structure determination, it does not provide dynamical information, and either experimental tricks or complementary experiments must be used to overcome the inherently static nature of crystallographic structures. Here we used specific x-ray damage during temperature-controlled crystallographic experiments at a third-generation synchrotron source to trigger and monitor (Shoot-and-Trap) structural changes putatively involved in an enzymatic reaction. In particular, a nonhydrolyzable substrate analogue of acetylcholinesterase, the "off-switch" at cholinergic synapses, was radiocleaved within the buried enzymatic active site. Subsequent product clearance, observed at 150 K but not at 100 K, indicated exit from the active site possibly via a "backdoor." The simple strategy described here is, in principle, applicable to any enzyme whose structure in complex with a substrate analogue is available and, therefore, could serve as a standard procedure in kinetic crystallography studies.
Shoot-and-Trap: use of specific x-ray damage to study structural protein dynamics by temperature-controlled cryo-crystallography., Colletier JP, Bourgeois D, Sanson B, Fournier D, Sussman JL, Silman I, Weik M, Proc Natl Acad Sci U S A. 2008 Aug 19;105(33):11742-7. Epub 2008 Aug 13. PMID:18701720
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.
OTMA is a nonhydrolyzable substrate analogue of AChE. Its hydrolyzis is impossible as OTMA possesses carbon instead ester oxygen of AChE natural substrate ACh. Similarly to ACh, OTMA covalently binds to the TcAChE Ser200 Oγ at the catalytic anionic subsite (CAS). At this subsite OTMA also interacts with Trp84, Phe330 (cation-π interactions); Glu199 (electrostatic interaction); Gly118, Gly119, and Ala201 (hydrogen bonds). Of note, OTMA binds not only at CAS, but also at peripheral anionic subsite (PAS). The second OTMA molecule interacts with Trp279, Tyr70 (cation-π interactions), and Tyr121 (weak hydrogen bond) at this subsite. Thus, this dual binding mode of OTMA with TcAChE (to CAS and PAS) could be prototypical for AChE bivalent inhibitors.
About this Structure
2VJA is a 2 chains structure of sequences from Torpedo californica. Full crystallographic information is available from OCA.
Reference
Colletier JP, Bourgeois D, Sanson B, Fournier D, Sussman JL, Silman I, Weik M. Shoot-and-Trap: use of specific x-ray damage to study structural protein dynamics by temperature-controlled cryo-crystallography. Proc Natl Acad Sci U S A. 2008 Aug 19;105(33):11742-7. Epub 2008 Aug 13. PMID:18701720
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