Structures of the “open” and “closed” state of trypanosomal triosephosphate isomerase, as observed in a new crystal form: Implications for the reaction mechanism

Noble, M.E.M.; Zeelen, J.P.; Wierenga, Rik K.

doi:10.1002/prot.340160402

Cited by 62 publications

(74 citation statements)

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“…et al, 1992; Noble et al, 1993;Falzone et al, 1994;Morton & Matthews, 1995). The loops presumably play an important role in gating ligand association and dissociation, but their energetic contributions to molecular recognition remain unclear.…”

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Thermodynamic and structural consequences of flexible loop deletion by circular permutation in the streptavidin‐biotin system

et al. 1998

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A circularly permuted streptavidin (CP5 1/46) has been designed to remove the flexible polypeptide loop that undergoes an open to closed conformational change when biotin is bound. The original termini have been joined by a tetrapeptide linker, and four loop residues have been removed, resulting in the creation of new N-and C-termini. Isothermal titration calorimetric studies show that the association constant has been reduced approximately six orders of magnitude below that of wild-type streptavidin to lo7 M-'. The AHo of biotin association for CP51/46 is reduced by 11.1 kcal/mol. Crystal structures of CP51/46 and its biotin complex show no significant alterations in the binding site upon removal of the loop. A hydrogen bond between Ser45 and Ser52 found in the absence of biotin is broken in the closed conformation as the side-chain hydroxyl of Ser45 moves to hydrogen bond to a ureido nitrogen of biotin. This is true in both the wild-type and CP5 1/46 forms of the protein, and the hydrogen bonding interaction might thus help nucleate closure of the loop. The reduced entropic cost of binding biotin to CP51/46 is consistent with the removal of this loop and a reduction in entropic costs associated with loop closure and immobilization. The reduced enthalpic contribution to the free energy of binding is not readily explainable in terms of the molecular structure, as the binding contacts are nearly entirely conserved, and only small differences in solvent accessible surfaces are observed relative to wild-type streptavidin.Keywords: circular permutation; ligand binding thermodynamics; loop deletion; molecular recognition; streptavidin; structure-based drug design 3V. Chu and S. Freitag should be considered co-first authors on this paper.Abbreviations: CP51/46, circularly permuted streptavidin with Glu5 1 as N-and Ala46 as C-terminus, respectively, and GGGS connecting amino acidsAla13 and Ser139; RMSD, root-mean-square distance; MPD, 2-methylpentane-2,4-diol. et al., 1992; Noble et al., Falzone et al., 1994; Morton & Matthews, 1995). The loops presumably play an important role in gating ligand association and dissociation, but their energetic contributions to molecular recognition remain unclear. The free energy of binding is the result of balancing the entropic costs/benefits of ordering of loops and release of bound water with the enthalpic benefits of burying nonpolar surface area and establishing bonding contacts. It is expected that loop folding may lead to energetic signatures similar to those associated with protein folding (Murphy et al., 1993;Spolar & Record, 1994).We are interested in defining the high-affinity reaction coordinate of streptavidin, where the conformational change of a flexible binding loop is a prominent feature accompanying biotin association (Hendrickson et al., 1989;Weber et al., 1989). High-affinity protein-ligand interactions play an important role in biology and often are the goal of structure-based drug design projects. The streptavidin-biotin affinity couple, with a K, of app...

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Thermodynamic and structural consequences of flexible loop deletion by circular permutation in the streptavidin‐biotin system

et al. 1998

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“…In the 4 different crystal structures the TIM subunit can be observed in 7 different crystallographic environments (Table 2). In Table 3 (Wierenga et al, 1991b) and the C2(big cell) crystals (Noble et al, 1993b) have been published previously.…”

Section: Resultsmentioning

confidence: 99%

“…The refinement of the crystal structures of the P212121 and C2(big cell) crystal forms have already been described (Wierenga et al, 1991b;Noble et al, 1993b). The crystals of the CZ(smal1 cell) and P1 form were grown from trypanosomal TIM purified according to previously described protocols (Borchert et al, 1993), using the hanging drop procedure.…”

Section: Methodsmentioning

confidence: 99%

“…Keywords: crystal contacts; DMSO; mutagenesis; flexible loop; TIM Crystal structures of trypanosomal triosephosphate isomerase (TIM) have already been described for 2 different crystal forms (Wierenga et al, 1991b;Noble et al, 1993b). The structures of TIM of chicken (Banner et al, 1975), yeast (Lolis et al, 1990, and Escherichia coli (Noble et al, 1993a) are also known.…”

Section: Introductionmentioning

confidence: 99%

“…The crystal contacts in the 4 different crystal forms are predominantly generated by polar residues in loops. A statistical analysis of the residues involved in crystal contacts shows that, in particular, serines are frequently involved in these interactions; 19% of the exposed serines are involved in crystal contacts.Keywords: crystal contacts; DMSO; mutagenesis; flexible loop; TIM Crystal structures of trypanosomal triosephosphate isomerase (TIM) have already been described for 2 different crystal forms (Wierenga et al, 1991b;Noble et al, 1993b). The structures of TIM of chicken (Banner et al, 1975), yeast (Lolis et al, 1990, and Escherichia coli (Noble et al, 1993a) are also known.…”

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Comparison of the structures and the crystal contacts of trypanosomal triosephosphate isomerase in four different crystal forms

et al. 1994

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Triosephosphate isomerase (TIM) is a dimeric enzyme consisting of 2 identical subunits. Trypanosoma1 TIM can be crystallized in 4 different spacegroups: P212121, C2(big cell), C2(small cell), and P1. The P1 crystal form only grows in the presence of 1.4 M DMSO; there are 2 DMSO binding sites per subunit. The structures have been refined at a resolution of 1.83 A, 2.10 A, 2.13 A, and 1.80 A, respectively. In the 4 different spacegroups the TIM subunit can be observed in the context of 7 different crystallographic environments. In the C2 cells, the dimer 2-fold axis coincides with a crystallographic 2-fold axis. The similarities and differences of the 7 subunits are discussed. In 6 subunits the flexible loop (loop 6) is open, whereas in the P21212, cell, the flexible loop of subunit 2 is in an almost closed conformation. The crystal contacts in the 4 different crystal forms are predominantly generated by polar residues in loops. A statistical analysis of the residues involved in crystal contacts shows that, in particular, serines are frequently involved in these interactions; 19% of the exposed serines are involved in crystal contacts.Keywords: crystal contacts; DMSO; mutagenesis; flexible loop; TIM Crystal structures of trypanosomal triosephosphate isomerase (TIM) have already been described for 2 different crystal forms (Wierenga et al., 1991b;Noble et al., 1993b). The structures of TIM of chicken (Banner et al., 1975), yeast (Lolis et al., 1990, and Escherichia coli (Noble et al., 1993a) are also known. TIM is a dimeric glycolytic enzyme, consisting of 2 identical subunits. Each subunit has the regular (@a)8-fold, with 8 parallel @-strands in the center, covered by 8 a-helices on the outside. The loops between the @-strands and the subsequent helices, referred to as loop 1-loop 8, are important for dimer formation and catalysis. A characteristic property of TIM is the conformational flexibility of loop 6, also referred to as the flexible loop. This loop has been observed in open (unliganded) and closed (liganded) conformations. There is a 7-A movement at the tip of this loop between the open and closed conformations (Joseph et al., 1990 (Wierenga et al., 1991b). The comparison (Wierenga et al., 1992) of the open and closed TIM structures has shown that the open structures of chicken, yeast, and trypanosomal TIM are essentially identical to each other; also the closed structures of yeast and trypanosoma1 TIM are essentially identical. In the open structures of TIM, the tip of loop 6 is sometimes seen to be highly flexible. Despite the structural heterogeneity associated with this flexibility, unliganded trypanosomal TIM can be crystallized readily into well-diffracting crystals, under different conditions and in different crystal forms. Previously the refined crystal structures of a P212121 form, crystallized in spacegroup P212121, and a C2 form, crystallized in spacegroup C2, have been discussed. The asymmetric unit of the P212121 form is 1 dimer. Due to the coincidence of dimer and crystallographic 2-f...

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Ab initio models for receptor-ligand interactions in proteins. 4. Model assembly study of the catalytic mechanism of triosephosphate isomerase

Peräkylä

Pakkanen

1996

Proteins

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The catalytic mechanism of triosephosphate isomerase (TIM) was investigated with ab initio quantum mechanical calculations. Electrostatic interactions between the quantum mechanical active site and the protein and solvent environment were modeled using the finite difference Poission‐Boltzman method. The complexes of TIM with the substrate dihydroxyacetone phosphate (DHAP), five possible intermediates and the product glyceraldehyde‐3‐phosphate (GAP) were optimized in the active‐site model at the 3‐21G(*) level and energy profile for the proton abstraction from DHAP by the active‐site Glu167 was calculated at the MP2/3‐21G(*)//3‐21G(*) level. Calculated energetics of the enzyme reaction were found to be in reasonable agreement with the experimental findings. Calculations revealed that an enediol of the substrate is a probable intermediate in the enzyme reaction. It was suggested that the proton abstracted from the substrate by the active‐site glutamate goes to the carbonyl oxygen of the substrate producing enediol intermediate either directly or after it is exchanged with solvent. © 1996 Wiley‐Liss, Inc.

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Structures of the “open” and “closed” state of trypanosomal triosephosphate isomerase, as observed in a new crystal form: Implications for the reaction mechanism

Cited by 62 publications

References 30 publications

Thermodynamic and structural consequences of flexible loop deletion by circular permutation in the streptavidin‐biotin system

Thermodynamic and structural consequences of flexible loop deletion by circular permutation in the streptavidin‐biotin system

Comparison of the structures and the crystal contacts of trypanosomal triosephosphate isomerase in four different crystal forms

Ab initio models for receptor-ligand interactions in proteins. 4. Model assembly study of the catalytic mechanism of triosephosphate isomerase

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