Identification of substrate binding site of GroEL minichaperone in solution.

It is difficult to obtain high-resolution structural information on the substrate-binding site of intact GroEL. But minichaperones, domains containing the peptide-binding site of GroEL, do constitute tractable systems for detailed studies. A peptide-binding site was located in crystals of a minichaperone and proposed to constitute a model for substrate-binding. We have now located the substrate binding site of the minichaperone GroEL(193-335) in solution by labelling it at various positions with a fluorescent probe and detecting which positions are perturbed on binding a denatured substrate. The fluorescence of a probe attached to a cysteine residue engineered at position 228 (N terminus of helix H8), 241 (helix H8), 261 (helix H9), or 267 (helix H9) was affected significantly by binding of substrate. But there was little change for a label at positions 193, 212, 217 or 293. The dissociation constants between substrates and minichaperone were evaluated from fluorescence anisotropy assays. The effects of salt and temperature were the same as those with intact GroEL. These results indicate that the region around helices H8 and H9 is the substrate-binding site for the apical domain fragment. Intriguingly, the same site is involved in the binding of GroES. Thus, an important function of GroES in the regulation of the activity of GroEL for substrates is to displace the bound substrate by competing for its binding site.     

GroEL recognises sequential and non-sequential linear structural motifs compatible with extended beta-strands and alpha-helices.

The chaperonin GroEL binds a variety of polypeptides that share no obvious sequence similarity. The precise structural, chemical and dynamic features that are recognised remain largely unknown. Structural models of the complex between GroEL and its co-chaperonin GroES, and of the isolated apical domain of GroEL (minichaperone; residues 191-376) with a 17 residue N-terminal tag show that a linear sequential sequence (extended beta-strand) can be bound. We have analysed characteristics of the motifs that bind to GroEL by using affinity panning of immobilised GroEL minichaperones for a library of bacteriophages that display the fungal cellulose-binding domain of the enzyme cellobiohydrolase I. This protein has seven non-sequential residues in its binding site that form a linear binding motif with similar dimensions and characteristics to the peptide tag that was bound to the minichaperone GroEL(191-376). The seven residues thus form a constrained scaffold. We find that GroEL does bind suitable mutants of these seven residues. The side-chains recognised do not have to be totally hydrophobic, but polar and positively charged chains can be accommodated. Further, the spatial distribution of the side-chains is also compatible with those in an alpha-helix. This implies that GroEL can bind a wide range of structures, from extended beta-strands and alpha-helices to folded states, with exposed side-chains. The binding site can accommodate substrates of approximately 18 residues when in a helical or seven when in an extended conformation. The data support two activities of GroEL: the ability to act as a temporary parking spot for sticky intermediates by binding many motifs; and an unfolding activity of GroEL by binding an extended sequential conformation of the substrate.     

NMR analysis of the binding of a rhodanese peptide to a minichaperone in solution.

A detailed structural analysis of interactions between denatured proteins and GroEL is essential for an understanding of its mechanism. Minichaperones constitute an excellent paradigm for obtaining high-resolution structural information about the binding site and conformation of substrates bound to GroEL, and are particularly suitable for NMR studies. Here, we used transferred nuclear Overhauser effects to study the interaction in solution between minichaperone GroEL(193-335) and a synthetic peptide (Rho), corresponding to the N-terminal alpha-helix (residues 11 to 23) of the mitochondrial rhodanese, a protein whose in vitro refolding is mediated by minichaperones. Using a 60 kDa maltose-binding protein (MBP)-GroEL(193-335) fusion protein to increase the sensitivity of the transferred NOEs, we observed characteristic sequential and mid-range transferred nuclear Overhauser effects. The peptide adopts an alpha-helical conformation upon binding to the minichaperone. Thus the binding site of GroEL is compatible with binding of alpha-helices as well as extended beta-strands. To locate the peptide-binding site on GroEL(193-335), we analysed changes in its chemical shifts on adding an excess of Rho peptide. All residues with significant chemical shift differences are localised in helices H8 and H9. Non-specific interactions were not observed. This indicates that the peptide Rho binds specifically to minichaperone GroEL(193-335). The binding region identified by NMR in solution agrees with crystallographic studies with small peptides and with fluorescence quenching studies with denatured proteins.     

From minichaperone to GroEL 3: properties of an active single-ring mutant of GroEL

The next step in our reductional analysis of GroEL was to study the activity of an isolated single seven-membered ring of the 14-mer. A known single-ring mutant, GroEL(SR1), contains four point mutations that prevent the formation of double-rings. That heptameric complex is functionally inactive because it is unable to release GroES. We found that the mutation E191G, which is responsible for the temperature sensitive (ts) Escherichia coli allele groEL44 and is located in the hinge region between the intermediate and apical domains of GroEL, appears to function by weakening the binding of GroES, without destabilizing the overall structure of GroEL44 mutant. We introduced, therefore, the mutation E191G into GroEL(SR1) in order to generate a single-ring mutant that may have weaker binding of GroES and hence be active. The new single-ring mutant, GroEL(SR44), was indeed effective in refolding both heat and dithiothreitol-denatured mitochondrial malate dehydrogenase with great efficiency. Further, unlike all smaller constructs of GroEL, the expression of GroEL(SR44) in E. coli that contained no endogenous GroEL restored biological viability, but not as efficiently as does wild-type GroEL. We envisage the notional evolution of the structure and properties of GroEL. The minichaperone core acts as a primitive chaperone by providing a binding surface for denatured states that prevents their self-aggregation. The assembly of seven minichaperones into a ring then enhances substrate binding by introducing avidity. The acquisition of binding sites for ATP then allows the modulation of substrate binding by introducing the allosteric mechanism that causes cycling between strong and weak binding sites. This is accompanied by the acquisition by the heptamer of the binding of GroES, which functions as a lid to the central cavity and competes for peptide binding sites. Finally, dimerization of the heptamer enhances its biological activity.     

Minichaperone (GroEL191-345) mediated folding of MalZ proceeds by binding and release of native and functional intermediates

The isolated apical domain of GroEL consisting of residues 191–345 (known as “minichaperone”) binds and assists the folding of a wide variety of client proteins without GroES and ATP, but the mechanism of its action is still unknown. In order to probe into the matter, we have examined minichaperone-mediated folding of a large aggregation prone protein Maltodextrin-glucosidase (MalZ). The key objective was to identify whether MalZ exists free in solution, or remains bound to, or cycling on and off the minichaperone during the refolding process. When GroES was introduced during refolding process, production of the native MalZ was inhibited. We also observed the same findings with a trap mutant of GroEL, which stably captures a predominantly non-native MalZ released from minichaperone during refolding process, but does not release it. Tryptophan and ANS fluorescence measurements indicated that refolded MalZ has the same structure as the native MalZ, but that its structure when bound to minichaperone is different. Surface plasmon resonance measurements provide an estimate for the equilibrium dissociation constant KD for the MalZ-minichaperone complex of 0.21 ± 0.04 μM, which are significantly higher than for most GroEL clients. This showed that minichaperone interacts loosely with MalZ to allow the protein to change its conformation and fold while bound during the refolding process. These observations suggest that the minichaperone works by carrying out repeated cycles of binding aggregation-prone protein MalZ in a relatively compact conformation and in a partially folded but active state, and releasing them to attempt to fold in solution.     

Probing the "annealing" mechanism of GroEL minichaperone using molecular dynamics simulations

Although the intact chaperonin machinery is needed to rescue natural substrate proteins (SPs) under non-permissive conditions the "minichaperone" alone, containing only the isolated apical domain of GroEL, can assist folding of a certain class of proteins. To understand the annealing function of the minichaperone, we have carried out molecular dynamics simulations in the NPT ensemble totaling 300ns for four systems; namely, the isolated strongly binding peptide (SBP), the minichaperone, and the SBP and a weakly binding peptide (WBP) in complex with the minichaperone. The SBP, which is structureless in isolation, adopts a beta-hairpin conformation in complex with the minichaperone suggesting that favorable non-specific interactions of the SPs confined to helices H and I of the apical domains can induce local secondary structures. Comparison of the dynamical fluctuations of the apo and the liganded forms of the minichaperone shows that the stability (needed for SP capture) involves favorable hydrophobic interactions and hydrogen bond network formation between the SBP and WBP, and helices H and I. The release of the SP, which is required for the annealing action, involves water-mediated interactions of the charged residues at the ends of H and I helices. The simulation results are consistent with a transient binding release (TBR) model for the annealing action of the minichaperone. According to the TBR model, SP annealing occurs in two stages. In the first stage the SP is captured by the apical domain. This is followed by SP release (by thermal fluctuations) that places it in a different region of the energy landscape from which it can partition rapidly to the native state with probability Phi or be trapped in another misfolded state. The process of binding and release can result in enhancement of the native state yield. The TBR model suggests "that any cofactor that can repeatedly bind and release SPs can be effective in assisting protein folding." By comparing the structures of the non-chaperone alpha-casein (which has no sequence similarity with the apical domain) and the minichaperone and the hydrophobicity profiles we show that alpha-casein has a pair of helices that have similar sequence and structural profiles as H and I. Based on this comparison we identify residues that stabilize (destabilize) alpha-casein-protein complexes. This suggests that alpha-casein assists folding by the TBR mechanism.     

The binding of bis-ANS to the isolated GroEL apical domain fragment induces the formation of a folding intermediate with increased hydrophobic surface not observed in tetradecameric GroEL

The extent of hydrophobic exposure upon bis-ANS binding to the functional apical domain fragment of GroEL, or minichaperone (residues 191-345), was investigated and compared with that of the GroEL tetradecamer. Although a total of seven molecules of bis-ANS bind cooperatively to this minichaperone, most of the hydrophobic sites were induced following initial binding of one to two molecules of probe. From the equilibrium and kinetics studies at low bis-ANS concentrations, it is evident that the native apical domain is converted to an intermediate conformation with increased hydrophobic surfaces. This intermediate binds additional bis-ANS molecules. Tyrosine fluorescence detected denaturation demonstrated that bis-ANS can destabilize the apical domain. The results from (i) bis-ANS titrations, (ii) urea denaturation studies in the presence and absence of bis-ANS, and (iii) intrinsic tyrosine fluorescence studies of the apical domain are consistent with a model in which bis-ANS binds tightly to the intermediate state, relatively weakly to the native state, and little to the denatured state. The results suggest that the conformational changes seen in apical domain fragments are not seen in the intact GroEL oligomer due to restrictions imposed by connections of the apical domain to the intermediate domain and suppression of movement due to quaternary structure.     

From minichaperone to GroEL 2: importance of avidity of the multisite ring structure

Structural studies on minichaperones and GroEL imply a continuous ring of binding sites around the neck of GroEL. To investigate the importance of this ring, we constructed an artificial heptameric assembly of minichaperones to mimic their arrangement in GroEL. The heptameric Gp31 co-chaperonin from bacteriophage T4, an analogue of GroES, was used as a scaffold to display the GroEL minichaperones. A fusion protein, MC(7), was generated by replacing a part of the highly mobile loop of Gp31 (residues 23-44) with the sequence of the minichaperone (residues 191-376 of GroEL). The purified recombinant protein assembled into a heptameric ring composed of seven 30.6 kDa subunits. Although single minichaperones (residues 193-335 to 191-376 of GroEL) have certain chaperone activities in vitro and in vivo, they cannot refold heat and dithiothreitol-denatured mitochondrial malate dehydrogenase (mtMDH), a reaction that normally requires GroEL, its co-chaperonin GroES and ATP. But, MC(7) refolded MDH in vitro. The expression of MC(7) complements in vivo two temperature-sensitive Escherichia coli alleles, groEL44 and groEL673, at 43 degrees C. Although MC(7) could not compensate for the complete absence of GroEL in vivo, it enhanced the colony-forming ability of cells containing limiting amounts of wild-type GroEL at 37 degrees C. MC(7 )also reduces aggregate formation and cell death in mammalian cell models of Huntington's disease. The assembly of seven minichaperone subunits on a heptameric ring significantly improves their activity, demonstrating the importance of avidity in GroEL function.     

Glu257 in GroEL is a sensor involved in coupling polypeptide substrate binding to stimulation of ATP hydrolysis

The ATPase activity of many types of molecular chaperones is stimulated by polypeptide substrate binding via molecular mechanisms that are, for the most part, unknown. Here, we report that such stimulation of the ATPase activity of GroEL is abolished when its conserved apical domain residue Glu257 is replaced by alanine. This mutation is also found to convert the ATPase profile of GroEL, a group I chaperonin, into one that is characteristic of group II chaperonins. Steady-state and transient kinetic analysis indicate that both effects are due, at least in part, to a reduction of the affinity of GroEL for ADP. This finding indicates that nonfolded proteins stimulate ATP hydrolysis by accelerating the off-rate of the ADP formed, thereby allowing more rapid cycles of ATP binding and hydrolysis.     

The crystal structure of spermidine/spermine N1-acetyltransferase in complex with spermine provides insights into substrate binding and catalysis

The enzyme spermidine/spermine N (1)-acetyltransferase (SSAT) catalyzes the transfer of acetyl groups from acetylcoenzyme A to spermidine and spermine, as part of a polyamine degradation pathway. This work describes the crystal structure of SSAT in complex with coenzyme A, with and without bound spermine. The complex with spermine provides a direct view of substrate binding by an SSAT and demonstrates structural plasticity near the active site of the enzyme. Associated water molecules bridge several of the intermolecular contacts between spermine and the enzyme and form a "proton wire" between the side chain of Glu92 and the N1 amine of spermine. A single water molecule can also be seen forming hydrogen bonds with the side chains of Glu92, Asp93, and the N4 amine of spermine. Site-directed mutation of Glu92 to glutamine had a detrimental effect on both substrate binding and catalysis and shifted the optimal pH for enzyme activity further into alkaline solution conditions, while mutation of Asp93 to asparagine affected both substrate binding and catalysis without changing the pH dependence of the enzyme. Considered together, the structural and kinetic data suggest that Glu92 functions as a catalytic base to drive an otherwise unfavorable deprotonation step at physiological pH.     

Role of two active site Glu residues in the molecular action of botulinum neurotoxin endopeptidase

Botulinum neurotoxin type A (BoNT/A) light chain (LC) is a zinc endopeptidase that causes neuroparalysis by blocking neurotransmitter release at the neuromuscular junctions. The X-ray crystal structure of the toxin reveals that His223 and His227 of the Zn(2+) binding motif HEXXH directly coordinate the active site zinc. Two Glu residues (Glu224 and Glu262) are also part of the active site, with Glu224 coordinating the zinc via a water molecule whereas Glu262 coordinates the zinc directly as the fourth ligand. In the past we have investigated the topographical role of Glu224 by replacing it with Asp thus reducing the side chain length by 1.4 A that reduced the endopeptidase activity dramatically [L. Li, T. Binz, H. Niemann, and B.R. Singh, Probing the role of glutamate residue in the zinc-binding motif of type A botulinum neurotoxin light chain, Biochemistry 39 (2000) 2399-2405]. In this study we have moved the Glu 224 laterally by a residue (HXEXH) to assess its positional influence on the endopeptidase activity, which was completely lost. The functional implication of Glu262 was investigated by replacing this residue with aspartate and glutamine using site-directed mutagenesis. Substitution of Glu262 with Asp resulted in a 3-fold decrease in catalytic efficiency. This mutation did not induce any significant structural alterations in the active site and did not interfere with substrate binding. Substitution of Glu262 with Gln however, dramatically impaired the enzymatic activity and this is accompanied by global alterations in the active site conformation in terms of topography of aromatic amino acid residues, zinc binding, and substrate binding, resulting from the weakened interaction between the active site zinc and Gln. These results suggest a pivotal role of the negatively charged carboxyl group of Glu262 which may play a critical role in enhancing the stability of the active site with strong interaction with zinc. The zinc may thus play structural role in addition to its catalytic role.     

Chaperone activity of a chimeric GroEL protein that can exist in a single or double ring form.

The molecular chaperone GroEL is a protein complex consisting of two rings each of seven identical subunits. It is thought to act by providing a cavity in which a protein substrate can fold into a form that has no propensity to aggregate. Substrate proteins are sequestered in the cavity while they fold, and prevented from diffusion out of the cavity by the action of the GroES complex, that caps the open end of the cavity. A key step in the mechanism of action of GroEL is the transmission of a conformational change between the two rings, induced by the binding of nucleotides to the GroEL ring opposite to the one containing the polypeptide substrate. This conformational change then leads to the discharge of GroES from GroEL, enabling polypeptide release. Single ring forms of GroEL are thus predicted to be unable to chaperone the folding of GroES-dependent substrates efficiently, since they are unable to discharge the bound GroES and unable to release folded protein. We describe here a detailed functional analysis of a chimeric GroEL protein, which we show to exist in solution in equilibrium between single and double ring forms. We demonstrate that whereas the double ring form of the GroEL chimera functions effectively in refolding of a GroES-dependent substrate, the single ring form does not. The single ring form of the chimera, however, is able to chaperone the folding of a substrate that does not require GroES for its efficient folding. We further demonstrate that the double ring structure of GroEL is likely to be required for its activity in vivo.     

Production of minichaperone (sht GroEL191-345) and its function in the refolding of recombinant human interferon gamma

The recombinant minichaperone sht GroEL191-345 was cultivated in a 3.7 L stirred bioreactor with the high yield of 216.2 mg/L broth. In the refolding of recombinant human interferon gamma (rhuIFN-gamma) inclusion bodies, more than 2-3 fold enhancement in protein mass recovery and total activity were observed in the presence of free or immobilized minichaperone to the refolding buffer.     

Protein substrate binding induces conformational changes in the chaperonin GroEL. A suggested mechanism for unfoldase activity

Chaperonins are molecules that assist proteins during folding and protect them from irreversible aggregation. We studied the chaperonin GroEL and its interaction with the enzyme human carbonic anhydrase II (HCA II), which induces unfolding of the enzyme. We focused on conformational changes that occur in GroEL during formation of the GroEL-HCA II complex. We measured the rate of GroEL cysteine reactivity toward iodo[2-(14)C]acetic acid and found that the cysteines become more accessible during binding of a cysteine free mutant of HCA II. Spin labeling of GroEL with N-(1-oxyl-2,2,5, 5-tetramethyl-3-pyrrolidinyl)iodoacetamide revealed that this additional binding occurred because buried cysteine residues become accessible during HCA II binding. In addition, a GroEL variant labeled with 6-iodoacetamidofluorescein exhibited decreased fluorescence anisotropy upon HCA II binding, which resembles the effect of GroES/ATP binding. Furthermore, by producing cysteine-modified GroEL with the spin label N-(1-oxyl-2,2,5, 5-tetramethyl-3-pyrrolidinyl)iodoacetamide and the fluorescent label 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid, we detected increases in spin-label mobility and fluorescence intensity in GroEL upon HCA II binding. Together, these results show that conformational changes occur in the chaperonin as a consequence of protein substrate binding. Together with previous results on the unfoldase activity of GroEL, we suggest that the chaperonin opens up as the substrate protein binds. This opening mechanism may induce stretching of the protein, which would account for reported unfoldase activity of GroEL and might explain how GroEL can actively chaperone proteins larger than HCA II.     

GroEL Recognizes an Amphipathic Helix and Binds to the Hydrophobic Side

GroEL is an essential Escherichia coli molecular chaperon that uses ATP to facilitate correct folding of a range of proteins in a cell. Central to the GroEL substrate diversity is how GroEL recognizes the substrates. The interaction between GroEL and substrate has been proposed to be largely hydrophobic because GroEL interacts with proteins in non-native conformations but not in native forms. Analysis of GroEL substrate proteins reveals that one of its main substrates are proteins with αβ folding domains, suggesting that GroEL may stabilize the collapsed αβ core by binding to hydrophobic surfaces that are usually buried between the α and β elements. In this study, we characterize the interaction between GroEL and a peptide derived from our previous selection via a phage display method. NMR studies map the peptide-binding site to the region containing Helices H and I, which is consistent with evidence that this region comprises the primary substrate-binding site. The peptide is largely unstructured in solution but adopts a helical conformation when bound to the GroEL apical domain with a moderate affinity (K_d = 17.1 ± 2.5 μm). The helical conformation aligns residues to form an amphipathic structure, and the hydrophobic side of this amphipathic helix interacts with GroEL as suggested by fluorescence quenching studies. Together with previous structural studies on the GroEL-peptide complexes, our work supports the notion that the amphipathic secondary elements in the substrate proteins may be the structural motif recognized by GroEL.The bacterial chaperonin GroEL and its co-chaperonin GroES are essential for cell viability by assisting folding of a wide range of proteins via an ATP-dependent mechanism (1–3). Structurally, fourteen 57-kDa GroEL subunits assemble into two back-to-back stacking heptameric rings, giving rise to two functionally independent central cavities (4). Each GroEL subunit folds into three distinctive domains: equatorial domain, intermediate domain, and apical domain. The equatorial domains contain the ATP-binding sites and provide most of the intra-ring interactions and all the inter-ring interactions. The apical domains form the rims of the central cavities and contain the binding sites for the substrate proteins and GroES. The intermediate domains link the apical domains and the equatorial domains. For the co-chaperonin GroES, seven GroES subunits, of 10 kDa each, assemble into a heptamer ring (5, 6). In forming the GroEL-GroES complex, GroES caps one end of GroEL, and large structural changes are observed in both GroEL and GroES (7). In GroEL, the apical domain is rotated 90° along its axis and 60° upwards, and the intermediate domain is closed down ∼25° to the equatorial domain. A loop in GroES (residues 17–33) that is unstructured in the isolated GroES adopts a β-turn structure and forms contact with the GroEL apical domain. Compared with the unliganded GroEL, the volume of the enclosed GroEL-GroES cavity is doubled, and the surface lining the wall of the GroEL cavity changes from hydrophobic to hydrophilic.A wealth of information derived from both intensive biochemical and structural characterizations has revealed a general role of GroEL-GroES in assisting protein folding (see reviews in Refs. 3, 8, and 9). Briefly, GroEL binds the substrate proteins in their aggregation-prone non-native states, preventing them from aggregating. Binding of ATP to the substrate occupied GroEL ring (cis-ring) presumably induces large conformational change in GroEL that promotes binding of GroES to the cis-ring. As a result of ATP and GroES binding, the substrate protein is displaced into the GroEL central cavity, initiating the folding process. Both hydrolysis of ATP in the cis-ring and binding of ATP to the substrate unoccupied ring (trans ring) weaken the GroES-GroEL interaction, and ATP binding to the trans ring results in the dissociation of GroES from GroEL, releasing substrate from the central cavity of GroEL. The released substrate may continue folding into the native state if in a folding competent state or may rebind to GroEL if it is still misfolded.One of the most intriguing aspects of the GroE-assisted folding is the substrate promiscuity. It has been shown that about 300 Escherichia coli proteins can interact with GroEL, and these proteins are diverse in terms of both structures and functions (10). A range of techniques have been applied to investigate this important yet complex aspect, and salient features regarding GroEL-substrate interactions have emerged. The apical domains, on the rim of the GroEL central cavity, contain the main substrate-binding site (11–13). Structural flexibility, reflected by both high temperature factors of the apical domain in the crystal structure of tetradecameric GroEL (14) and conformational multiplicity around Helix H and I (15), is proposed to account for the diverse spectrum of GroEL substrates. Mutational studies on GroEL suggest that the GroEL-substrate interactions are largely hydrophobic (16). Structural study on GroEL-substrate interaction, however, is hindered mainly because of the multiple conformations of the bound substrate protein. Very recently, NMR techniques have been used to directly investigate the bound conformations of the substrate (17, 18); yet the nature of GroEL-substrate interaction is not revealed. Peptides may mimic segments of substrate proteins, and studies of GroEL-peptide interactions have uncovered detailed intermolecular interactions and provided insights into principles of substrate recognition by GroEL. The bound peptides may adopt α-helix (19–23), β-hairpin (15), or extended conformations (24), and despite different conformations, they all appear to bind to Helix H and I of GroEL. Hydrophobic interaction dominates the interface between GroEL and peptides in either β-hairpin or extended structures and is proposed so between GroEL and α-helical peptides. These detailed structural characterizations on GroEL-peptide interactions have contributed to dissecting the complex nature of the substrate recognition by GroEL (25).We previously identified a high affinity peptide (strong binding peptide (SBP))² for GroEL using a phage display method and found that SBP adopts a β-hairpin structure bound to GroEL (15, 26). To investigate the contribution of the β-turn in SBP to the GroEL-SBP interaction, we have created various SBP variants with the intension to disrupt the β-turn structure and have studied their binding to GroEL. One of the peptides (termed SBP-W2DP6V), however, adopts a helical conformation when bound to GroEL by NMR analysis. NMR results also map the peptide-binding site on GroEL to be a region formed by Helix H and I. The helical peptide has an amphipathic feature, and fluorescence studies provide direct evidence that the hydrophobic face is involved in the interaction with GroEL. Our structural analysis, combined with previous studies, suggests that GroEL recognizes the amphipathic property in the secondary structures of the substrate protein and binds preferably to the hydrophobic side of these structural elements to stabilize and preserve their structures.     

Flexibility of GroES Mobile Loop Is Required for Efficient Chaperonin Function

Chaperonin GroEL and its partner GroES assist the folding of nascent and stress-damaged proteins in an ATP-dependent manner. Free GroES has a flexible "mobile loop" and binds to GroEL through the residues at the tip of the loop, capping the central cavity of GroEL to provide the substrate polypeptide a cage for secure in-cage folding. Here, we show that restriction of the flexibility of the loop by a disulfide cross-linking between cysteines within the loop results in the inefficient formation of a stable GroEL-polypeptide-GroES ternary complex and inefficient folding. Then, we generated substrate proteins with enhanced binding affinity to GroEL by fusion of one or two SBP (strongly binding peptide for GroEL) sequences and examined the effect of disulfide cross-linking on the assisted folding. The results indicate that the higher the binding affinity of the substrate polypeptide to GroEL, the greater the contribution of the mobile loop flexibility to efficient in-cage folding. It is likely that the flexibility helps GroES capture GroEL's binding sites that are already occupied by the substrate polypeptide with various binding modes.     

The crystal structure of phosphinothricin in the active site of glutamine synthetase illuminates the mechanism of enzymatic inhibition

Phosphinothricin is a potent inhibitor of the enzyme glutamine synthetase (GS). The resolution of the native structure of GS from Salmonella typhimurium has been extended to 2.5 A resolution, and the improved model is used to determine the structure of phosphinothricin complexed to GS by difference Fourier methods. The structure suggests a noncovalent, dead-end mechanism of inhibition. Phosphinothricin occupies the glutamate substrate pocket and stabilizes the Glu327 flap in a position which blocks the glutamate entrance to the active site, trapping the inhibitor on the enzyme. One oxygen of the phosphinyl group of phosphinothricin appears to be protonated, because of its proximity to the carboxylate group of Glu327. The other phosphinyl oxygen protrudes into the negatively charged binding pocket for the substrate ammonium, disrupting that pocket. The distribution of charges in the glutamate binding pocket is complementary to those of phosphinothricin. The presence of a second ammonium binding site within the active site is confirmed by its analogue thallous ion, marking the ammonium site and its protein ligands. The inhibition of GS by methionine sulfoximine can be explained by the same mechanism. These models of inhibited GS further illuminate its catalytic mechanism.     

Direct evidence for a glutamate switch necessary for substrate recognition: crystal structures of lysine epsilon-aminotransferase (Rv3290c) from Mycobacterium tuberculosis H37Rv

Lysine epsilon-aminotransferase (LAT) is a PLP-dependent enzyme that is highly up-regulated in nutrient-starved tuberculosis models. It catalyzes an overall reaction involving the transfer of the epsilon-amino group of L-lysine to alpha-ketoglutarate to yield L-glutamate and alpha-aminoadipate-delta-semialdehyde. We have cloned and characterized the enzyme from Mycobacterium tuberculosisH37Rv. We report here the crystal structures of the enzyme, the first from any source, in the unliganded form, external aldimine with L-lysine, with bound PMP and with its C5 substrate alpha-ketoglutarate. In addition to interaction details in the active site, the structures reveal a Glu243 "switch" through which the enzyme changes substrate specificities. The unique substrate L-lysine is recognized specifically when Glu243 maintains a salt-bridge with Arg422. On the other hand, the binding of the common C5 substrates L-glutamate and alpha-ketoglutarate is enabled when Glu243 switches away and unshields Arg422. The structures reported here, sequence conservation and earlier mutational studies suggest that the "glutamate switch" is an elegant solution devised by a subgroup of fold type I aminotransferases for recognition of structurally diverse substrates in the same binding site and provides for reaction specificity.     

Annealing function of GroEL: structural and bioinformatic analysis

The Escherichia coli chaperonin system, GroEL–GroES, facilitates folding of substrate proteins (SPs) that are otherwise destined to aggregate. The iterative annealing mechanism suggests that the allostery-driven GroEL transitions leading to changes in the microenvironment of the SP constitutes the annealing action of chaperonins. To describe the molecular basis for the changes in the nature of SP–GroEL interactions we use the crystal structures of GroEL (T state), GroEL–ATP (R state) and the GroEL–GroES–(ADP)₇ (R″ state) complex to determine the residue-specific changes in the accessible surface area and the number of tertiary contacts as a result of the T→R→R″ transitions. We find large changes in the accessible area in many residues in the apical domain, but relatively smaller changes are associated with residues in the equatorial domain. In the course of the T→R transition the microenvironment of the SP changes which suggests that GroEL is an annealing machine even without GroES. This is reflected in the exposure of Glu386 which loses six contacts in the T→R transition. We also evaluate the conservation of residues that participate in the various chaperonin functions. Multiple sequence alignments and chemical sequence entropy calculations reveal that, to a large extent, only the chemical identities and not the residues themselves important for the nominal functions (peptide binding, nucleotide binding, GroES and substrate protein release) are strongly conserved. Using chemical sequence entropy, which is computed by classifying aminoacids into four types (hydrophobic, polar, positively charged and negatively charged) we make several new predictions that are relevant for peptide binding and annealing function of GroEL. We identify a number of conserved peptide binding sites in the apical domain which coincide with those found in the 1.7 Å crystal structure of ‘mini-chaperone’ complexed with the N-terminal tag. Correlated mutations in the HSP60 family, that might control allostery in GroEL, are also strongly conserved. Most importantly, we find that charged solvent-exposed residues in the T state (Lys 226, Glu 252 and Asp 253) are strongly conserved. This leads to the prediction that mutating these residues, that control the annealing function of the SP, can decrease the efficacy of the chaperonin function.     

ATP-triggered conformational changes delineate substrate-binding and -folding mechanics of the GroEL chaperonin

The chaperonin GroEL assists the folding of nascent or stress-denatured polypeptides by actions of binding and encapsulation. ATP binding initiates a series of conformational changes triggering the association of the cochaperonin GroES, followed by further large movements that eject the substrate polypeptide from hydrophobic binding sites into a GroES-capped, hydrophilic folding chamber. We used cryo-electron microscopy, statistical analysis, and flexible fitting to resolve a set of distinct GroEL-ATP conformations that can be ordered into a trajectory of domain rotation and elevation. The initial conformations are likely to be the ones that capture polypeptide substrate. Then the binding domains extend radially to separate from each other but maintain their binding surfaces facing the cavity, potentially exerting mechanical force upon kinetically trapped, misfolded substrates. The extended conformation also provides a potential docking site for GroES, to trigger the final, 100° domain rotation constituting the "power stroke" that ejects substrate into the folding chamber.