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BioMedNet Journal Collection known wild-type structures. The dimer-interface interactions cause L1 and L4 to be very rigid, as is clear from the B-factor plot of wtTIM (Fig. 5). In the structures of monomeric TIMs, Lys13 is either mobile, or has adopted structures with a negative (unstrained) value for [in monoTIM-SS and monoTIM-W(2PG)], indicating that the dimer -interface interactions are required to stabilize the strained conformation of Lys13 in the wild-type structure. The increased flexibility of loops L1, L4 and L8 in monoTIM could be due to the shortening of L3 alone, but is probably caused by the monomerization itself. The first possibility does not seem very likely, because the changed residues of wild -type loop L3 (residues 68–82; [6] [8]) do not interact with any atom of L1 or L4 of the same subunit. In wtTIM, L6, together with the adjacent loops L5 and L7, can be in an open or closed state. In the monomeric TIM structures, the open structure is observed for monoTIM-SS and the closed structure is observed for monoTIM-SS(PGH), monoTIM-SS(2PG) and monoTIM in complex with sulphate. Clearly, the open/closed mechanism still operates in monomeric TIM and in the closed form the catalytic glutamate, Glu167, at the beginning of L6, adopts the same conformation as in wtTIM. What is different in monomeric TIM is the flexibility of loops L1, L4 and L8. Loop L8 is only different from wtTIM in the monoTIM-SS structure and loop L4 is only different in the monoTIM structure (Fig. 4a). The structure of monoTIM-W(2PG) shows that both Lys13 (L1) and His95 (L4) can adopt conformations similar to those seen in wtTIM (Fig. 7). In wild type, Lys13 is important for binding the substrate [15]. The exact role of Lys13 in the catalysis has not yet been established [16] [17], but mutational analysis has shown that any positively charged side chain at this position permits some catalytic activity, demonstrating the importance of the overall electrostatic properties of the activesite pocket [15]. His95 is important for the proton shuffling between the two oxygen atoms of the substrate [17] [18] [19]. The structures of monoTIM-SS(PGH) and monoTIM-W(2PG) indicate that the side chains of Lys13 and His95 can play the same role in catalysis as in wild type. Apart from the increased flexibility of the front loops, at least one other difference exists between the active sites of monoTIM and wtTIM that will also influence the catalytic efficiency. In wtTIM, the active-site pocket is shielded from bulk solvent by the other subunit, but in monoTIM the active site is much more exposed to bulk solvent, and this will affect its dielectric properties. The three different monoTIMs have very similar catalytic properties despite the structural differences seen in the various crystal forms. This is consistent with the conclusion that the active-site loops are mobile in solution, and that the loop conformations seen in the crystal structures are induced by the crystallization conditions. We might imagine two kinds of mobility in solution: firstly, fast switches between two (or more) discrete conformational states (as might be the case for L4) and secondly, 'continuous flexibility' (as discussed for L1). Without performing experiments in solution (e.g. using NMR), it is hard to discriminate between these two possibilities at the present time. Biological implications Triosephosphate isomerase (TIM) is a dimeric enzyme. Each subunit consists of eight ( ) units connected by loops. The loops following the -strands (loops L1–8) are the so-called 'front' loops. The dimer is stabilized by tight interactions between the residues of the four dimer-interface loops, L1–4. Two important catalytic residues, Lys13 (on L1) and His95 (on L4), reside on these dimerinterface loops. In this study we have analyzed the structural properties of these loops in TIMs engineered to form stable monomers (monoTIMs). In the wild-type (wt) TIM dimer these loops are very rigid, but in our comparison of four crystal structures of monoTIMs these loops are seen to exhibit remarkable conformational flexibility. Nevertheless, the monoTIMs have residual catalytic activity. This apparent paradox is explained by our observation that the catalytic residues on these loops can adopt conformations similar, although not identical, to those in wtTIM, in the presence of active-site ligands. http://journals.bmn.com/journals/list/browse?uid=JSTR.st3702&node=TOC%40%40JSTR%4003%40t07%4003_t07 Page 18 of 25 9/11/01
BioMedNet Journal Collection The flexibility of loops L1, L4 and L8 in monoTIM implies that in wild-type subunits, free in solution, these loops are also flexible. The sequence of the monoTIMs differ from wtTIM by the deletion of seven residues from the interface loop, L3. It is very likely that the observed conformational flexibility of loops L1, L4 and L8 in monoTIM is caused by the absence of the other subunit rather than the deletion itself. If this is the case, then the assembly of unfolded wtTIM monomers into dimers follows a path, in agreement with a consecutive folding-association mechanism [20]: firstly, the monomers adopt a folded structure, in which the interface loops are still mobile (as inferred from these studies); secondly, these monomers recognize each other and subsequently undergo further structural reshuffling, leading to the final tertiary and quaternary structure. Further characterization of pointmutation variants of wtTIM, engineered to make dimerization impossible, will be required to confirm this hypothesis. The flexibility of some of the front loops in monoTIM has the following implication for designing monoTIMs with increased catalytic activity. Sequence changes in loops L1 and L4 (or their immediate environments) which increase their rigidity and hence allow the active-site residues to adopt wild - type conformations, should enhance the catalytic activity of monoTIM. Protein design experiments targeting loop L1 have been initiated. Materials and methods DNA technology, protein expression and protein purification E. coli strains XL1-Blue [21] and BL21(DE3) [22] were used as hosts for the plasmids throughout the genetic manipulations and the expression of the proteins, respectively. The plasmids encoding the variant proteins were, except for the mutations in the TIM gene, identical to plasmid pTIM described previously [23]. For monoTIM-SS, serine codons were introduced by site-directed mutagenesis at positions 45 and 46 in the monoTIM gene, located on plasmid pTIM [23]. MonoTIM-W is derived from monoTIM by changing the alanine at position 100 into a tryptophan. The site-directed mutagenesis was done by the polymerase chain reaction (PCR) using the overlap-extension procedure [24]. The DNA sequences of the cloned PCR-amplified fragments were verified. MonoTIM-SS and monoTIM-W expression and purification to homogeneity were carried out as described for monoTIM [6]. The kinetic constants were measured in a coupled enzyme assay as previously described [7]. Crystallization and data collection The monoTIM-SS and monoTIM-W crystals were grown at room temperature using the hanging drop method. The crystallization conditions for monoTIM-SS, monoTIM-SS(PGH), and monoTIM-W(2PG) crystals (Table 1) were found by a standard screening procedure [25]. One crystal was used for each dataset. The data were collected at room temperature with a FAST area detector, mounted on a rotating anode. Data processing was performed with MADNES [26]. The data collection statistics are summarized in Table 2. <strong>Structure</strong> determination and refinement MonoTIM-SS: The structure was solved by molecular replacement using the CCP4 program package [27]. The self-rotation function (10–4 Å) gave a very clear peak for a local twofold axis. As a search model for the molecular replacement calculations subunit -1 of the 1.83 Å refined structure of trypanosomal TIM [5TIM in the Brookhaven Protein Data Bank (PDB)] was used, after omitting the residues of loops L1, L3, L4, and L6. The molecular replacement calculations gave a clear indication of the orientation and position of the two monoTIM-SS molecules. The starting model was rebuilt in a 3 Å map using O [28], and refined with X-PLOR [29] first at 3 Å and subsequently at higher http://journals.bmn.com/journals/list/browse?uid=JSTR.st3702&node=TOC%40%40JSTR%4003%40t07%4003_t07 Page 19 of 25 9/11/01
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