Refined 1.83 Å structure of trypanosomal triosephosphate isomerase crystallized in the presence of 2.4 m-ammonium sulphate : A comparison with the structure of the trypanosomal triosephosphate isomerase-glycerol-3-phosphate complex

Abstract

Triosephosphate isomerase (TIM) is a dimeric glycolytic enzyme. TIM from Trypanosoma brucei brucei has been crystallized at pH 7.0 in 2.4 m-ammonium sulphate. The well-diffracting crystals have one dimer per asymmetric unit. The structure has been refined at 1.83 Å resolution with an R-factor of 18.3% for all data between 6 Å and 1.83 Å (37,568 reflections). The model consists of 3778 protein atoms and 297 solvent atoms. Subunit 1 is involved in considerably more crystal contacts than subunit 2. Correlated with these differences in crystal packing is the observation that only in the active site of subunit 2 is a sulphate ion bound. Furthermore, significant differences with respect to structure and flexibility are observed in three loops near the active site. In particular, there is a 7 Å positional difference of the tip of the flexible loop (loop 6) when comparing subunit 1 and subunit 2. Also, the neighbouring loops (loop 5 and loop 7) have significantly different conformations and flexibility. In subunit 1, loop 6 is in an “open” conformation, in subunit 2, loop 6 is in an “almost closed” conformation. Only in the presence of a phosphate-containing ligand, such as glycerol-3-phosphate, does loop 6 take up the “closed” conformation. Loop 6 and loop 7 (and also to some extent loop 5) are rather flexible in the almost closed conformation, but well defined in the open and closed conformations. The closing of loop 6 (167 to 180), as observed in the almost closed conformation, slightly changes the main-chain conformation of the catalytic glutamate, Glu167, leading to a change of the χ 1 angle of this residue from ~−60 ° to ~60 ° and the weakening of the hydrogen bonds between its polar side-chain atoms and Ser96. In the closed conformation, in the presence of glycerol-3-phosphate, the main-chain atoms of Glu167 remain in the same position as in the almost closed conformation, but the side-chain has rotated around the CACB bond changing χ 1 from ∼60 ° to ∼−60 °. In this new position the hydrogen bonding to Ser96 is completely lost and also a water-mediated salt bridge between OE2(Glu167) and NE(Arg99) is lost. Comparison of the two independently refined subunits, showed that the root-mean-square deviation for all 249 CA atoms is 0.9 Å; for the CA atoms of the β-strands this is only 0.2 Å. The average B-factor for all subunit 1 and subunit 2 atoms is 20 Å 2 and 25 Å 2, respectively. This difference can be understood because subunit 1 is involved in many more crystal contacts than subunit 2. The correlation coefficient between the B-factor values of subunit 1 and subunit 2 is 0.7, despite the difference in flexibility of some loops. There are 279 water molecules; 25 water molecules are completely buried inside the protein. A subset of water molecules (approximately 100) consists of equivalent water molecules in subunit 1 and subunit 2: 50 water molecules contacting subunit 1 can be superimposed (within 0.5 Å) by the local dyad on water molecules near subunit 2. The interface residues in trypanosomal TIM are rather different from other TIMs. In addition, one of the interface loops, loop 3, is one residue shorter. In trypanosomal TIM there are two hydrophobic patches at the interface. In addition, there are polar interactions: salt bridges as well as non-charged hydrogen bonds. About 50% of the hydrogen bonds at the interface are water-mediated hydrogen bonds. At the interface there are 18 completely buried water molecules, nine of these water molecules are hydrogen-bonded to both subunits. These 18 buried water molecules are grouped in seven water clusters, consisting of one to five water molecules.