Dynamic Requirements for a Functional Protein Hinge

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Abstract

The enzyme triosephosphate isomerase (TIM) is a model of catalytic efficiency. The 11 residue loop 6 at the TIM active site plays a major role in this enzymatic prowess. The loop moves between open and closed states, which facilitate substrate access and catalysis, respectively. The N and C-terminal hinges of loop 6 control this motion. Here, we detail flexibility requirements for hinges in a comparative solution NMR study of wild-type (WT) TIM and a quintuple mutant (PGG/GGG). The latter contained glycine substitutions in the N-terminal hinge at Val167 and Trp168, which follow the essential Pro166, and in the C-terminal hinge at Lys174, Thr175, and Ala176. Previous work demonstrated that PGG/GGG has a tenfold higher Km value and 103-fold reduced kcat relative to WT with either d-glyceraldehyde 3-phosphate or dihyrdroxyacetone phosphate as substrate. Our NMR results explain this in terms of altered loop-6 dynamics in PGG/GGG. In the mutant, loop 6 exhibits conformational heterogeneity with corresponding motional rates < 750 s 1 that are an order of magnitude slower than the natural WT loop 6 motion. At the same time, nanosecond timescale motions of loop 6 are greatly enhanced in the mutant relative to WT. These differences from WT behavior occur in both apo PGG/GGG and in the form bound to the reaction-intermediate analog, 2-phosphoglycolate (2-PGA). In addition, as indicated by 1H, 15N and 13CO chemical-shifts, the glycine substitutions diminished the enzyme's response to ligand, and induced structural perturbations in apo and 2-PGA-bound forms of TIM that are atypical of WT. These data show that PGG/GGG exists in multiple conformations that are not fully competent for ligand binding or catalysis. These experiments elucidate an important principle of catalytic hinge design in proteins: structural rigidity is essential for focused motional freedom of active-site loops.

Introduction

Enzymes make extensive use of conformational changes throughout their catalytic cycle that are, in many cases, essential to their function. Triosephosphate isomerase (TIM, EC 5.3.1.1) is an important case in which motion plays a significant role in the rate-limiting catalytic step. TIM is a very efficient and faithful catalyst of the interconversion (Scheme 1) between dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (GAP), enabling isomerization near the diffusion-limited rate,1 while limiting formation of the toxic side product, methylglyoxal, to only one molecule per 105 catalytic cycles.2 A critical aspect of TIM catalysis is the participation of a highly conserved active-site Ω loop, the 11 residue loop 6, which consists of three residue N and C-terminal hinges and an intervening five residue tip (Scheme 2). Ω loops typically reside on the surface of proteins and consist of 6–20 amino acid residues with a short distance (≤ 10 Å) between the N and C-terminal residues, yielding a resemblance to the Greek letter Ω.3., 4.

In TIM, loop 6 plays a functional role via its motion between two major conformational states: open and closed (Figure 1). In the open conformation, the substrate has ready access to both the active site and the bulk solvent. The closed conformation is observed in X-ray crystallographic studies in which ligand is bound in the TIM active site. Closure of loop 6 involves an approximately 7 Å movement at its tip (Cα of Thr172). This closed form is stabilized by hydrogen bonds between the amide NH of loop 6 residue Ala176 and the hydroxyl group of Tyr208 within loop 7 (residues 208–211) and between the Ala176 carbonyl group and the Ser211 hydroxyl group†. Notably, loop 6 closure is accompanied by significant structural changes in loop 7 that allow repositioning of the carboxylate group of Glu165, which is the catalytic base, into its functionally competent position.5., 6., 7. In addition, the central five residues of loop 6 have been shown to be important for preventing unwanted production of methylglyoxal,2 yet the only direct contact of loop 6 with the ligand is a single hydrogen bond between the ligand phosphate group and the backbone NH of Gly171. This stabilization of an intermediate state and the possible coordination of loop 6 motion with that of loop 7 and Glu165 facilitate the enzymatic reaction, and physically allow passage of the ligand between the active site and solvent. Thus, motion of loop 6 is essential to overall function in TIM.

Several lines of evidence support the notion that loop 6 motion is limiting to the reaction rate in the direction of the DHAP to GAP interconversion,1 each placing loop motion of the order of 103–104 s 1 in yeast (Saccharomyces cerevisiae) TIM. Computational studies have long suggested that loop 6 moves on a microsecond timescale with rate-limiting effect.8., 9. Solid-state NMR relaxation studies10., 11. of 2H-labeled Trp168 and solution-state NMR studies12 of 19F-labeled Trp168 also indicate that loop 6 moves at a rate of 104 s 1 and in a manner that is likely rate-limiting to catalysis. Furthermore, these rates were confirmed by temperature-jump relaxation spectroscopy utilizing Trp168 fluorescence,13 and supported by a study revealing that conformational motion on this timescale enhances 15N spin-relaxation rates within loop 6 and other sites vicinal to it.14., 15. In addition, the solid-state10 and solution-state14., 15. NMR experiments indicate that loop 6 moves in both apo and bound enzyme forms and, therefore, that loop motion is not ligand-gated.

The interconversion between opened and closed conformations of loop 6 is a rigid-body motion in which only residues in the N and C-terminal hinges experience changes in backbone dihedral angles.16., 17. Hydrogen bonds involving loop 6 residues are another prominent feature of the open-close motion. Upon closure, the amide NH of Gly171 in the tip of the loop comes within 2.8 Å of the O3 oxygen atom in the substrate phosphate, whereas the NH of Ala176 in the C-terminal hinge forms a critical hydrogen bond with the Tyr208 hydroxyl in loop 7.8., 18., 19. Upon closure, tight intra-loop hydrogen bonding also occurs between the amide groups of Ala169 and Ile170 in the loop 6 center and carbonyl groups of Pro166 and Val167 in the N-terminal hinge, respectively. Not surprisingly, residues in loop 6 are highly conserved in over 130 TIM sequences as described,20 and as investigated by genetic,21., 22. kinetic23., 24. and crystallographic20 means. In the loop 6 center, sequence conservation is driven by a structure that: (1) encapsulates the active site during catalysis; (2) facilitates the Gly171 backbone hydrogen bond to substrate and the intra-enzyme hydrogen bonds of Ala169 and Ile170; and (3) positions the Ile170 side-chain to shield the substrate–enzyme interaction from bulk solvent. In the hinge regions of loop 6, conservation reflects: (1) the backbone hydrogen-bonding constraints at Pro166, Val167 and Ala176; and (2) the requirements for functional loop 6 motion.

Packing is a prominent factor for both of the loop 6 hinges, as depicted in Figure 2. Analysis of these interactions provides insight into significant features relevant to their conformational motion. For the first two residues (C1 and C2) of the C-terminal hinge,21 the side-chains are loosely positioned and only require compatibility with solvent exposure in both the open and closed conformations. In contrast, the third residue (C3) is selected for important packing interactions in the apo (on-average open) form of the loop. The open conformation requires the C3 side-chain to fit into a small hydrophobic pocket or else destabilize the open form, thus blocking access to the active site. This explains the evolutionary preference for alanine at C3. Meanwhile, among the three N-terminal hinge sites (N1–N3), the PXW sequence is dominant (with 98% X = I/V/L).20 This consensus is likely driven: (1) by structural and dynamic constraints on the catalytic Glu165 at position (N1–1); and (2) by packing constraints on the N1 and N3 sites and, to a lesser extent, on N2.22 It has been noted that the few observed deviations of the N-hinge from PXW retain the N1 proline and are correlated with changes in the otherwise strictly conserved YGGS motif of loop 7.20

Sampson and co-workers investigated the requirements for functional hinge sequences in TIM using genetic library approaches.21., 22., 23. These studies revealed that some C-hinge sequence variability can be tolerated in the context of catalytic activity, while the amino acid sequence requirements at the N-terminal hinge are much more stringent. An additional result from these studies was the interdependence of the N and C-terminal hinge sequences, which results in non-additive effects on catalytic activity. Curiously, these library experiments did not yield a single semi-active mutant (> 10% of wild-type activity) with glycine in the N-terminal hinge. Likewise, only four of 86 semi-active C-hinge mutants included a glycine substitution (none with multiple glycine residues). Furthermore, glycine is not observed at any hinge position in any of the naturally occurring TIM enzymes sequenced to date. All of this is in spite of the fact that glycine does not conflict with any of the noted factors influencing hinge conservation and design: glycine substitution would not sterically destabilize the open or closed forms of loop 6, nor would it alter the capability for backbone hydrogen bonding at the C3 and N1 and N2 hinge sites. A remaining possibility is that dynamic factors necessitate selection against glycine; namely, that, in spite of the motional role of the hinges, proper enzyme function requires that they exhibit a degree of rigidity not provided by glycine, the smallest of the amino acids.

To explore this hypothesis, Xiang et al.24 characterized glycine-containing hinge mutants of chicken TIM (cTIM): PGG, with hinge sequences P166/V167G/W168G at the N-terminal hinge, GGG with K174G/T175G/A176G mutations the C-terminal hinge, and PGG/GGG, the combination of the N and C-hinge mutants. Pro166 was always retained due to the absolute conservation at N1. Biochemical characterization revealed a 200-fold reduction in kcat for the PGG and GGG mutations, and a 2500-fold drop in kcat for PGG/GGG. The substrate Km value for all three mutants was elevated tenfold relative to WT, indicating equivalent reductions of affinity for substrate. Additionally, 31P NMR spectroscopy of the reaction intermediate analog, 2-phosphoglycolate (2-PGA) (Scheme 1) indicated that, when bound to PGG/GGG, the phosphorus moiety experiences a solvent-like environment unlike that observed in WT. In spite of these differences from WT, none of the mutants exhibited an increase in the rate of methylglyoxal production, indicating that loop 6 does not prematurely open (or fail to close) during catalysis. Furthermore, each mutant exhibits primary deuterium kinetic isotope effects in the DHAP to GAP direction similar to that of WT, suggesting that enolization is partially rate-limiting regardless of mutation.24 Altogether, these data suggest that the decreased enzymatic activity of PGG/GGG is a result of relatively infrequent closure of loop 6 and continued, if diminished, coupling of that motion with rearrangement of the catalytic base into its competent position.

The action of loop 6 appears to require a carefully balanced combination of motional freedom and structural rigidity that demands further study to understand its functional role. Here, we use solution NMR experiments to investigate the dynamics of loop 6 for WT and PGG/GGG cTIM in the apo and 2-PGA-bound forms, which are the on-average opened and closed WT conformations, respectively. These studies illuminate the significant glycine-induced alteration of loop dynamics and provide convincing evidence for conformational heterogeneity and increased configurational entropy in the mutant. Finally, our analysis of biochemical data in the context of these NMR results allows construction of a kinetic model that explains the large drop in PGG/GGG catalytic activity via the presence of additional equilibria that yield multiple non-productive conformations, E* and E#S, of the apo enzyme and enzyme substrate complex, respectively.

Section snippets

Evaluation of hinge packing in loop 6

Much of the conservation of amino acid types in loop 6, and particularly at its hinge sites, is due to functional constraints on van der Waals packing, hydrogen bonding, and backbone dynamics. Figure 2 shows the packing interactions of the C and N-terminal hinge side-chains for both open and closed forms of the protein. In the C-terminal hinge,21 sites C1 and C2 must be compatible with solvent exposure in both the open and closed conformations. This is depicted in Figure 2(a)–(d) using apo and

Materials and Methods

Chemical reagents were purchased from Sigma (St. Louis, MO) unless noted otherwise.

Acknowledgements

J.P.L. acknowledges funding from the NIH (R01 GM070823) and support from an Alfred P. Sloan Foundation fellowship. J.G.K. completed this work with support from an NIH Kirchstein Postdoctoral Fellowship (F32 GM-66599-03). N.S.S. acknowledges support by an AC grant from the American Chemical Society-Petroleum Research Fund. N.S.S. is a member of the New York Structural Biology Center (NYSBC) supported by NIH grants GM066354 and RR017528 and New York State. We thank Dr Kaushik Dutta of the NYSBC

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    Present addresses: J. G. Kempf, Department of Chemistry, Rensselaer Polytechnic Institute, Troy, New York 12180, USA; J. Jung, Department of Genetic Medicine, Weill Medical College of Cornell University, New York, NY 10021, USA.

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