W0021

Rabbit Muscle Apo TIM Structure: A New Paradigm for the Active Site Loop Conformation. Ricardo Aparicio^1,3, Sérgio T. Ferreira² and Igor Polikarpov³, ¹Instituto de Física Gleb Wataghin, UNICAMP; ²Depto. de Bioquímica Médica, UFRJ and Laboratório Nacional de Luz Síncrotron; ³Instituto de Física de São Carlos, USP. Brazil. Email: ipolikarpov@if.sc.usp.br .

The active site loop of triose phosphate isomerase (TIM) exhibits a hinged-lid motion, alternating between the two well defined open and closed conformations. Until now the closed conformation had only been observed in holo structures of complexes with substrate analogues. Here we present the first rabbit muscle apo TIM structure, refined to 1.5 Å, in which the active site loop is either in the open or in the closed conformation in different subunits. Unexpectedly, chemical interactions characteristic for holo TIM structures are observed in the closed conformation for the apo structure described here. Additives used during crystallization (DMSO and TRIS molecules and Mg atoms) were modeled in the electron density maps. However, no specific binding of these molecules is observed in the active site or lid loop regions. To further investigate this unusual apo enzyme closed conformation, two more rabbit muscle TIM structures, one in the same and another in a different crystal form, were determined. They confirmed that the observed closed conformation can not be explained by crystal contacts. To rationalize why the active site loop is closed in the absence of ligand in one of the subunits, extensive comparison with previously solved TIM structures was carried out, supported by the bulk of available experimental information about enzyme kinetics and reaction mechanism of TIM. The simultaneous observation of both open and closed lid loop conformations in the chemically homogeneous TIM population might be related to a persistent conformational heterogeneity of TIM in solution. Moreover, our results strongly support the hypothesis that TIM active site loop movement is not ligand-gated.

RA gratefully ackowledges Drs. A. M. Brzozowski, E. J. Dodson and G. N. Murshudov (YSBL, UK). Financial support: FAPESP (RA and IP); FAPERJ and Howard Hughes Medical Institute (STF).