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PEDS Advance Access published online on May 27, 2004
Protein Engineering Design and Selection, doi:10.1093/protein/gzh048
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Received May 17, 2004
Accepted May 19, 2004

Article

Understanding protein lids: structural analysis of active hinge mutants in triosephosphate isomerase

I. Kursula 1, M. Salin 2, J. Sun 3, B. V. Norledge 2, A. M. Haapalainen 2, N. S. Sampson 3, R. K. Wierenga 2*

1 University of Oulu, Department of Biochemistry, Finland; Biocenter Oulu, P.O. Box 3000, FIN-90014 University of Oulu, Finland; European Molecular Biology Laboratory, Hamburg Outstation, c/o DESY, Notkestrasse 85, Building 25A, D-22603 Hamburg, Germany
2 University of Oulu, Department of Biochemistry, Finland; Biocenter Oulu, P.O. Box 3000, FIN-90014 University of Oulu, Finland
3 Department of Chemistry, State University of New York, Stony Brook, NY 11794-3400, USA

* To whom correspondence should be addressed. E-mail: Rik.Wierenga{at}oulu.fi.


  Abstract

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 and Sampson 1999, Biochemistry 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, while others (KVA, NSS) are essentially identical to wild type. The structural characterization of these six variants is reported. No significant structural differences compared to 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.

Keywords: Archae, evolution, flexible loop, structure, TIM


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