Evidence of Domain Movement in Thymidine Phosphorylase. Matthew J. Pugmire,^a & Steven E. Ealick^b, Department of Chemistry, Cornell University, Ithaca, New York 14853,^a Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853^b

Two new crystal forms of e. coli thymidine phosphorylase have been found; a monoclinic form (P2₁) and an orthorhombic form (I222). These structures have been solved and compared to the previously determined tetragonal form (P4₃2₁2) (Walter et al., 1990). Comparing the three refined structures provides evidence of domain movement of the a (residues 1-65, and 163-193) and a/[beta] (residues 80-154, and 197-440) domains, which is thought to be critical for enzymatic activity by closing the active sight cleft, and provides insight on how this movement might occur. Three hinge regions that allow the a and a/[beta]-domains to move relative to each other are identified. The monoclinic model appears to be the most open whereas the tetragonal model is the most closed where the a and a/[beta] domains have moved toward each other partially closing the active site cleft. Changes in the structure due to phosphate binding are identified including the formation of a hydrogen bond between His-119 and Gly-208 which helps to order the 115-120 loop that is disordered prior to phosphate binding. The formation of this hydrogen bond also appears to play a key role in the domain movement. The a-domain appears to move as a rigid body while the a/[beta]-domain has some slight non-rigid body movement that is associated with the formation of the His-119 - Gly-208 hydrogen bond. The relative motions of the domains between the three structures have been quantitatively described by calculating effective screw axes. Other flexible regions of the protein and residues that may be involved in domain movement are discussed.

Walter, M. R., Cook, W. J., Cole, L. B., Short, S. A., Koszalka, G. W., Krenitsky, T. A., & Ealick, S. E. (1990) J. Biol. Chem. 265, 14016-14022.