PEDS Advance Access originally published online on May 27, 2004
Protein Engineering Design and Selection 2004 17(4):375-382; doi:10.1093/protein/gzh048
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Understanding protein lids: structural analysis of active hinge mutants in triosephosphate isomerase
1Department of Biochemistry and Biocenter Oulu, University of Oulu, PO Box 3000, FIN-90014 University of Oulu, Finland, 2European Molecular Biology Laboratory, Hamburg Outstation, c/o DESY, Notkestrasse 85, Building 25A, D-22603 Hamburg, Germany and 3Department of Chemistry, State University of New York, Stony Brook, NY 11794-3400, USA 4Present address: Pharmaceutical Chemistry, University of California, San Francisco, CA 94143-2280, USA 5Present address: The Scripps Research Institute, La Jolla, CA 92037, USA
6 To whom correspondence should be addressed. E-mail: rik.wierenga{at}oulu.fi
The conformational switch from open to closed of the flexible loop 6 of triosephosphate isomerase (TIM) is essential for the catalytic properties of TIM. Using a directed evolution approach, active variants of chicken TIM with a mutated C-terminal hinge tripeptide of loop 6 have been generated (Sun,J. and Sampson,N.S., Biochemistry, 1999, 38, 11474–11481). In chicken TIM, the wild-type C-terminal hinge tripeptide is KTA. Detailed enzymological characterization of six variants showed that some of these (LWA, NPN, YSL, KTK) have decreased catalytic efficiency, whereas others (KVA, NSS) are essentially identical with wild-type. The structural characterization of these six variants is reported. No significant structural differences compared with the wild-type are found for KVA, NSS and LWA, but substantial structural adaptations are seen for NPN, YSL and KTK. These structural differences can be understood from the buried position of the alanine side chain in the C-hinge position 3 in the open conformation of wild-type loop 6. Replacement of this alanine with a bulky side chain causes the closed conformation to be favored, which correlates with the decreased catalytic efficiency of these variants. The structural context of loop 6 and loop 7 and their sequence conservation in 133 wild-type sequences is also discussed.
Received April 26, 2004; accepted May 19, 2004.
Edited by Alan Fersht
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