The allosteric transition in DnaK probed by infrared difference spectroscopy. Concerted ATP-induced rearrangement of the substrate binding domain

The biological activity of DnaK, the bacterial representative of the Hsp70 protein family, is regulated by the allosteric interaction between its nucleotide and peptide substrate binding domains. Despite the importance of the nucleotide-induced cycling of DnaK between substrate-accepting and releasing states, the heterotropic allosteric mechanism remains as yet undefined. To further characterize this mechanism, the nucleotide-induced absorbance changes in the vibrational spectrum of wild-type DnaK was characterized. To assign the conformation sensitive absorption bands, two deletion mutants (one lacking the C-terminal alpha-helical subdomain and another comprising only the N-terminal ATPase domain), and a single-point DnaK mutant (T199A) with strongly reduced ATPase activity, were investigated by time-resolved infrared difference spectroscopy combined with the use of caged-nucleotides. The results indicate that (1) ATP, but not ADP, binding promotes a conformational change in both subdomains of the peptide binding domain that can be individually resolved; (2) these conformational changes are kinetically coupled, most likely to ensure a decrease in the affinity of DnaK for peptide substrates and a concomitant displacement of the lid away from the peptide binding site that would promote efficient diffusion of the released peptide to the medium; and (3) the alpha-helical subdomain contributes to stabilize the interdomain interface against the thermal challenge and allows bidirectional transmission of the allosteric signal between the ATPase and substrate binding domains at stress temperatures (42 degrees C).     

Coupling between substrate binding and allosteric regulation in ribozyme catalysis

The contribution of substrate binding to allosteric regulation in the ribozyme catalysis has been investigated using allosteric ribozymes consisting of the hammerhead ribozyme and a flavin mononucleotide (FMN) aptamer. Kinetic parameters were measured for various lengths of the substrates with a wide range of binding energy. The maximum cleavage rate of each ribozyme was retained with the long substrates. However, the cleavage rates largely decreased by the truncation of the substrates according to loss in the free energy of substrate binding. The high sensitivity to the substrate lengths is attributed to the increase in the energetic requirement for the catalytic core folding, which is caused by the incorporation of the aptamer region. One role of FMN binding is assisting the promotion of the core folding through the stabilization of the aptamer domain. The allosteric effect is significantly expressed only when the substrate binding energy is insufficient for the core folding of the ribozyme-substrate complex. This type of allosteric interaction dominates the substrate dependency of another type of regulation. These results demonstrate that an adequate correlation between the type of regulation and the substrate binding is responsible for the effective allosteric interaction in the kinetic process.     

Effect of the polypeptide binding on the thermodynamic stability of the substrate binding domain of the DnaK chaperone

The effect of polypeptide binding on the stability of the substrate binding domain of the molecular chaperone DnaK has been studied by thermodynamic analysis. The calorimetric scan of the fragment of the substrate binding domain DnaK384-638, consisting of a beta-domain and an alpha-helical lid, showed two transitions centered at 56.2 and 76.0 degrees C. On the other hand, the thermal unfolding of the shorter fragment DnaK386-561, which lacks half of the alpha-helical lid, exhibited a single transition at 57.0 degrees C. Therefore, the transition of DnaK384-638 at 56.2 degrees C is mainly attributed to the unfolding of the beta-domain. The calorimetric scan of DnaK384-638D526N showed that the unfolding of the beta-domain was composed of two transitions. The polypeptide bound DnaK384-638 exhibited a symmetrical DSC peak at 58.6 degrees C, indicating that the substrate binding shifts the beta-domain toward a single cooperative unit. A low concentration of GdnHCl (<1.0 M) induced a conformational change in the beta-domain of DnaK384-638 without changes in the secondary structure. While the thermal unfolding of the beta-domain of DnaK384-638 was composed of two transitions in the presence of GdnHCl, the beta-domain of the substrate bound DnaK384-638 exhibited a single symmetrical DSC peak in the same condition. All together, our results indicate that complex between DnaK384-638 and substrate forms a rigid conformation in the beta-domain.     

A conformational transition involved in antagonistic substrate binding to the allosteric phosphofructokinase from Escherichia coli.

The binding of fructose 6-phosphate, ATP or its nonhydrolyzable analogue adenylyl 5'-(beta,gamma-methylenediphosphonate), ADP, and phosphoenolpyruvate to Escherichia coli phosphofructokinase has been studied by changes in the protein fluorescence and/or equilibrium dialysis. The results lead to the following conclusions: (1) tetrameric phosphofructokinase can bind four ATP but only two fructose-6-phosphate, and this binding occurs without cooperativity; (2) only two conformational states, T and R, with respectively a high and a low fluorescence, seem accessible to phosphofructokinase, which exists as a mixture of one-third R and two-third T states in the absence of ligand; (3) the substrate fructose 6-phosphate and the allosteric activator ADP bind preferentially to the low-fluorescence R state, while the other substrate, ATP [or its nonhydrolyzable analogue adenylyl 5'-(beta,gamma-methylenediphosphonate)], and the allosteric inhibitor phosphoenolpyruvate bind to the high-fluorescence T state; (4) the binding of a given ligand is cooperative, with a Hill coefficient of 2, only when this binding is accompanied by a complete shift from one state to the other; for instance, the binding of the ATP analogue adenylyl 5'-(beta,gamma-methylenediphosphonate) to the T state is cooperative only in the presence of fructose 6-phosphate which favors the R state. This behavior is qualitatively consistent with a concerted transition, but quite different from that described earlier for phosphofructokinase from steady-state activity measurements (Blangy et al., 1968). This discrepancy suggests that the allosteric properties of phosphofructokinase are due in part to ligand binding and in part to the kinetics of the enzymatic reaction.     

Structural insights into substrate binding by the molecular chaperone DnaK

How substrate affinity is modulated by nucleotide binding remains a fundamental, unanswered question in the study of 70 kDa heat shock protein (Hsp70) molecular chaperones. We find here that the Escherichia coli Hsp70, DnaK, lacking the entire alpha-helical domain, DnaK(1-507), retains the ability to support lambda phage replication in vivo and to pass information from the nucleotide binding domain to the substrate binding domain, and vice versa, in vitro. We determined the NMR solution structure of the corresponding substrate binding domain, DnaK(393-507), without substrate, and assessed the impact of substrate binding. Without bound substrate, loop L3,4 and strand beta3 are in significantly different conformations than observed in previous structures of the bound DnaK substrate binding domain, leading to occlusion of the substrate binding site. Upon substrate binding, the beta-domain shifts towards the structure seen in earlier X-ray and NMR structures. Taken together, our results suggest that conformational changes in the beta-domain itself contribute to the mechanism by which nucleotide binding modulates substrate binding affinity.     

The role of dynamics in allosteric regulation

The biomolecular conformational changes often associated with allostery are, by definition, dynamic processes. Recent publications have disclosed the role of pre-existing equilibria of conformational substates in this process. In addition, the role of dynamics as an entropic carrier of free energy of allostery has been investigated. Recent work thus shows that dynamics is pivotal to allostery, and that it constitutes much more than just the move from the 'T'-state to the 'R'-state. Emerging computational studies have described the actual pathways of allosteric change.     

Proteolytic fragments of insulysin (IDE) retain substrate binding but lose allosteric regulation

Treatment of an N-terminal-containing His6-tagged insulysin (His6-IDE) with proteinase K led to the initial cleavage of the His tag and linker region. This was followed by C-terminal cleavages resulting in intermediate fragments of approximately 95 and approximately 76 kDa and finally a relatively stable approximately 56 kDa fragment. The approximately 76 and approximately 56 kDa fragments exhibited a low level of catalytic activity but retained the ability to bind the substrate with a similar affinity as the native enzyme. The kinetics of the reaction of the IDE approximately 76 and approximately 56 kDa proteolytic fragments with a synthetic fluorogenic substrate produced hyperbolic substrate versus velocity curves, rather than the sigmoidal curve obtained with His6-IDE. The approximately 76 and approximately 56 kDa IDE proteolytic fragments were active toward the physiological peptides beta-endorphin, insulin, and amyloid beta peptide 1-40. Although activity was reduced by a factor of approximately 103-104 with these substrates, the relative activity and the cleavage sites were unchanged. Both the approximately 76 and approximately 56 kDa fragments retained the regulatory cationic binding site that binds ATP. Thus, the two proteinase K cleavage fragments of IDE retain the substrate- and ATP-binding sites but have low catalytic activity and lose the allosteric kinetic behavior of IDE. These data suggest a role of the C-terminal region of IDE in allosteric regulation.     

Mutations in the substrate binding domain of the Escherichia coli 70 kDa molecular chaperone, DnaK, which alter substrate affinity or interdomain coupling

In Escherichia coli, DnaK is essential for the replication of bacteriophage lambda DNA; this in vivo activity provides the basis of a screen for mutations affecting DnaK function. Mn PCR was used to introduce mutations into residues 405-468 of the C-terminal polypeptide-binding domain of DnaK. These mutant proteins were screened for the ability to propagate bacteriophage lambda in the background of a dnaK deficient cell line, BB1553. This initial screen identified several proteins which were mutant at multiple positions. The multiple mutants were further dissected into single mutants which remained negative for lambda propagation. Four of these single-site mutants were purified and assayed for biochemical functionality. Two single-site mutations, F426S and S427P, are localized in the peptide binding site and display weakened peptide binding affinity. This indicates that the crystallographically determined peptide binding site is also critical for in vivo lambda replication. Two other mutations, K414I and N451K, are located at the edge of the beta-sandwich domain near alpha-helix A. The K414I mutant binds peptide moderately well, yet displays defects in allosteric functions, including peptide-stimulated ATPase activity, ATP-induced changes in tryptophan fluorescence, ATP-induced peptide release, and elevated ATPase activity. The K414 position is close in tertiary structure to the linker region to the ATPase domain and reflects a specific area of the peptide-binding domain which is necessary for interdomain coupling. The mutant N451K displays defects in both peptide binding and allosteric interaction.     

NMR study of nucleotide-induced changes in the nucleotide binding domain of Thermus thermophilus Hsp70 chaperone DnaK: implications for the allosteric mechanism

We present an NMR investigation of the nucleotide-dependent conformational properties of a 44-kDa nucleotide binding domain (NBD) of an Hsp70 protein. Conformational changes driven by ATP binding and hydrolysis in the N-terminal NBD are believed to allosterically regulate substrate affinity in the C-terminal substrate binding domain. Several crystal structures of Hsc70 NBDs in different nucleotide states have, however, not shown significant structural differences. We have previously reported the NMR assignments of the backbone resonances of the NBD of the bacterial Hsp70 homologue Thermus thermophilus DnaK in the ADP-bound state. In this study we show, by assigning the NBD with the ATP/transition state analogue, ADP.AlFx, bound, that it closely mimics the ATP-bound state. Chemical shift difference mapping of the two nucleotide states identified differences in a cluster of residues at the interface between subdomains 1A and 1B. Further analysis of the spectra revealed that the ATP state exhibited a single conformation, whereas the ADP state was in slow conformational exchange between a form similar to the ATP state and another state unique to the ADP-bound form. A model is proposed of the allosteric mechanism based on the nucleotide state altering the balance of a dynamic equilibrium between the open and closed states. The observed chemical shift perturbations were concentrated in an area close to a previously described J-domain binding channel, confirming the importance of that region in the allosteric mechanism.     

Essential function of the built-in lid in the allosteric regulation of eukaryotic and archaeal chaperonins

Chaperonins are allosteric double-ring ATPases that mediate cellular protein folding. ATP binding and hydrolysis control opening and closing of the central chaperonin chamber, which transiently provides a protected environment for protein folding. During evolution, two strategies to close the chaperonin chamber have emerged. Archaeal and eukaryotic group II chaperonins contain a built-in lid, whereas bacterial chaperonins use a ring-shaped cofactor as a detachable lid. Here we show that the built-in lid is an allosteric regulator of group II chaperonins, which helps synchronize the subunits within one ring and, to our surprise, also influences inter-ring communication. The lid is dispensable for substrate binding and ATP hydrolysis, but is required for productive substrate folding. These regulatory functions of the lid may serve to allow the symmetrical chaperonins to function as 'two-stroke' motors and may also provide a timer for substrate encapsulation within the closed chamber.     

Topology and dynamics of the 10 kDa C-terminal domain of DnaK in solution

Hsp70 molecular chaperones contain three distinct structural domains, a 44 kDa N-terminal ATPase domain, a 17 kDa peptide-binding domain, and a 10 kDa C-terminal domain. The ATPase and peptide binding domains are conserved in sequence and are functionally well characterized. The function of the 10 kDa variable C-terminal domain is less well understood. We have characterized the secondary structure and dynamics of the C-terminal domain from the Escherichia coli Hsp70, DnaK, in solution by high-resolution NMR. The domain was shown to be comprised of a rigid structure consisting of four helices and a flexible C-terminal subdomain of approximately 33 amino acids. The mobility of the flexible region is maintained in the context of the full-length protein and does not appear to be modulated by the nucleotide state. The flexibility of this region appears to be a conserved feature of Hsp70 architecture and may have important functional implications. We also developed a method to analyze 15N nuclear spin relaxation data, which allows us to extract amide bond vector directions relative to a unique diffusion axis. The extracted angles and rotational correlation times indicate that the helices form an elongated, bundle-like structure in solution.     

Roles of the N domain of the AAA+ Lon protease in substrate recognition,allosteric regulation and chaperone activity

Degron binding regulates the activities of the AAA+ Lon protease in addition to targeting proteins for degradation. The sul20 degron from the cell‐division inhibitor SulA is shown here to bind to the N domain of Escherichia coli Lon, and the recognition site is identified by cross‐linking and scanning for mutations that prevent sul20‐peptide binding. These N‐domain mutations limit the rates of proteolysis of model sul20‐tagged substrates and ATP hydrolysis by an allosteric mechanism. Lon inactivation of SulA in vivo requires binding to the N domain and robust ATP hydrolysis but does not require degradation or translocation into the proteolytic chamber. Lon‐mediated relief of proteotoxic stress and protein aggregation in vivo can also occur without degradation but is not dependent on robust ATP hydrolysis. In combination, these results demonstrate that Lon can function as a protease or a chaperone and reveal that some of its ATP‐dependent biological activities do not require translocation.     

Ionic regulation of glutamate binding sites

Cl- and Ca2+ increase glutamate binding to rat synaptic plasma membranes (SPMs) by revealing a distinct class of L-glutamate (L-Glu) binding sites. The present study was conducted to examine both the anion specificity of this response and the nature of the interaction between Cl- and Ca2+. Of the anions tested, Br- was the most effective in increasing the levels of L-Glu binding. Other effective anions were Cl-, NO3- and formate while F-, HCO3-CIO4-, propionate, SO42- and PO43- were ineffective. The anion specificity was similar to that observed for the Cl- membrane channel, suggesting that this binding site and the ion channel may be related. In the absence of Cl-, Ca2+ has little effect on L-Glu binding. Increasing the Cl- concentration increased the apparent affinity (decreased KCa2+) of the Ca2+-stimulated, L-Glu binding component and also increased the maximal amount of the enhancement. Conversely, increasing Ca2+ levels increased the maximal enhancement of L-Glu binding brought about by Cl- without affecting the KCl- of the effect. Prior incubation of membranes with Ca2+ did not raise the level of L-Glu binding. Furthermore, EGTA was able to reverse the stimulation of L-Glu binding due to Ca2+. The results indicate that Ca2+ acts ionically to enhance L-Glu binding to rat SPMs.     

Catalytic activation of human glucokinase by substrate binding: residue contacts involved in the binding of D-glucose to the super-open form and conformational transitions

alpha-D-Glucose activates glucokinase (EC 2.7.1.1) on its binding to the active site by inducing a global hysteretic conformational change. Using intrinsic tryptophan fluorescence as a probe on the alpha-D-glucose induced conformational changes in the pancreatic isoform 1 of human glucokinase, key residues involved in the process were identified by site-directed mutagenesis. Single-site W-->F mutations enabled the assignment of the fluorescence enhancement (DeltaF/F(0)) mainly to W99 and W167 in flexible loop structures, but the biphasic time course of DeltaF/F(0) is variably influenced by all tryptophan residues. The human glucokinase-alpha-D-glucose association (K(d) = 4.8 +/- 0.1 mm at 25 degrees C) is driven by a favourable entropy change (DeltaS = 150 +/- 10 J.mol(-1).K(-1)). Although X-ray crystallographic studies have revealed the alpha-d-glucose binding residues in the closed state, the contact residues that make essential contributions to its binding to the super-open conformation remain unidentified. In the present study, we combined functional mutagenesis with structural dynamic analyses to identify residue contacts involved in the initial binding of alpha-d-glucose and conformational transitions. The mutations N204A, D205A or E256A/K in the L-domain resulted in enzyme forms that did not bind alpha-D-glucose at 200 mm and were essentially catalytically inactive. Our data support a molecular dynamic model in which a concerted binding of alpha-D-glucose to N204, N231 and E256 in the super-open conformation induces local torsional stresses at N204/D205 propagating towards a closed conformation, involving structural changes in the highly flexible interdomain connecting region II (R192-N204), helix 5 (V181-R191), helix 6 (D205-Y215) and the C-terminal helix 17 (R447-K460).     

GrpE N-terminal domain contributes to the interaction with Dnak and modulates the dynamics of the chaperone substrate binding domain

GrpE acts as a nucleotide exchange factor for DnaK, the main Hsp70 protein in bacteria, accelerating ADP/ATP exchange by several orders of magnitude. GrpE is a homodimer, each subunit containing three structural domains: a N-terminal unordered segment, two long coils and a C-terminal globular domain formed by a four-helix bundle, and a β-subdomain. GrpE association to DnaK nucleotide-binding domain involves side-chain and backbone interactions located within the “headpiece” of the cochaperone, which consists of the C-terminal half of the coils, the four-helix bundle and the β-subdomain. However, the role of the GrpE N-terminal region in the interaction with DnaK and the activity of the cochaperone remain controversial. In this study we explore the contribution of this domain to the binding reaction, using the wild-type proteins, two deletion mutants of GrpE (GrpE_34-197 and GrpE_69-197) and the isolated DnaK nucleotide-binding domain. Analysis of the thermodynamic binding parameters obtained by isothermal titration calorimetry shows that both GrpE N-terminal segments, 1-33 and 34-68, contribute to the binding reaction. Partial proteolysis and substrate dissociation kinetics also suggest that the N-terminal half of GrpE coils (residues 34-68) interacts with DnaK interdomain linker, regulates the nucleotide exchange activity of the cochaperone and is required to stabilize DnaK-substrate complexes in the ADP-bound conformation.     

Anti-peptide monoclonal antibody imaging of a common binding domain involved in muscle regulation.

Multiple-component regulatory protein systems function through a generalized mechanism where a single regulatory protein or ligand binds to a variety of receptors to modulate specific functions in a physiologically sensitive context. Muscle contraction is regulated by the interaction of actin with troponin I (TnI) or myosin in a Ca(2+)-sensitive manner. Actin utilizes a single binding domain (residues 1-28) to bind to residues 104-115 of TnI (Van Eyk JE, Sönnichsen FD, Sykes BD, Hodges RS, 1991, In: Rüegg JC, ed, Peptides as probes in muscle research, Heidelberg, Germany: Springer-Verlag, pp 15-31) and to myosin subfragment 1 (S1, an enzymatic fragment of myosin containing both the actin and ATP binding sites) (Van Eyk JE, Hodges RS, 1991, Biochemistry 30:11676-11682) in a Ca(2+)-sensitive manner. We have utilized an anti-TnI peptide (104-115) monoclonal antibody, Mab B4, that binds specifically to TnI, to image the common binding domain of actin and thus mimic the activity of actin including activation of the S1 ATPase activity and TnI-mediated regulation of the S1 ATPase. Mab B4 has also been utilized to identify a receptor binding domain on myosin (residues 633-644) that is recognized by actin. Interestingly, Mab B4 binds to the native protein receptors TnI and S1 with relative affinities of 100- and 25,000-fold higher than the binding affinity to the 12-residue peptide immunogen. Thus, anti-peptide monoclonal antibodies prepared against a receptor binding domain can mimic the ligand binding domain and be utilized as a powerful tool for the detailed analysis of complex multiple-component regulatory systems.     

Interactive binding between the substrate and allosteric sites of carbamoyl-phosphate synthetase

The interaction between Escherichia coli carbamoyl-phosphate synthetase (CPS) and a fluorescent analogue of an allosteric effector molecule, 1,N6-ethenoadenosine 5'-monophosphate (epsilon-AMP), has been detected by using fluorescence techniques and kinetic measurements. From fluorescence anisotropy titrations, it was found that epsilon-AMP binds to a single site on CPS with Kd = 0.033 mM. The nucleotide had a small activating effect on the rate of synthesis of carbamoyl phosphate but had no effect on the Km for ATP. To test whether epsilon-AMP binds to an allosteric site, allosteric effectors (UMP, IMP, and CMP), known to bind at the UMP/IMP site, were added to solutions containing the epsilon-AMP-CPS complex. With addition of these effector molecules, a progressive decrease of the fluorescence anisotropy was observed, indicating that bound epsilon-AMP was displaced by the allosteric effectors examined. From these titrations, the dissociation constants for UMP, IMP, CMP, ribose 5-phosphate, 2-deoxyribose 5-phosphate, and orthophosphate were determined. When MgATP, a substrate, was employed as a titrant, the observed decrease in anisotropy was consistent with the formation of a ternary complex (epsilon-AMP-CPS-MgATP). The effect of ATP binding, monitored at the allosteric site, was magnesium dependent, and free magnesium in solution was required to obtain a hyperbolic binding isotherm. Solvent accessibility of epsilon-AMP in binary (epsilon-AMP-CPS) and ternary (epsilon-AMP-CPS-MgATP) complexes was determined from acrylamide quenching, showing that the base of epsilon-AMP is well shielded from the solvent even in the presence of MgATP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural mechanism of allosteric substrate specificity regulation in a ribonucleotide reductase

Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides into deoxyribonucleotides, which constitute the precursor pools used for DNA synthesis and repair. Imbalances in these pools increase mutational rates and are detrimental to the cell. Balanced precursor pools are maintained primarily through the regulation of the RNR substrate specificity. Here, the molecular mechanism of the allosteric substrate specificity regulation is revealed through the structures of a dimeric coenzyme B12-dependent RNR from Thermotoga maritima, both in complexes with four effector-substrate nucleotide pairs and in three complexes with only effector. The mechanism is based on the flexibility of loop 2, a key structural element, which forms a bridge between the specificity effector and substrate nucleotides. Substrate specificity is achieved as different effectors and their cognate substrates stabilize specific discrete loop 2 conformations. The mechanism of substrate specificity regulation is probably general for most class I and class II RNRs.     

Regulation of N-cadherin dynamics at neuronal contacts by ligand binding and cytoskeletal coupling

N-cadherin plays a key role in axonal outgrowth and synaptogenesis, but how neurons initiate and remodel N-cadherin-based adhesions remains unclear. We addressed this issue with a semiartificial system consisting of N-cadherin coated microspheres adhering to cultured neurons transfected for N-cadherin-GFP. Using optical tweezers, we show that growth cones are particularly reactive to N-cadherin coated microspheres, which they capture in a few seconds and drag rearward. Such strong coupling requires an intact connection between N-cadherin receptors and catenins. As they move to the basis of growth cones, microspheres slow down while gradually accumulating N-cadherin-GFP, demonstrating a clear delay between bead coupling to the actin flow and receptor recruitment. Using FRAP and photoactivation, N-cadherin receptors at bead-to-cell contacts were found to continuously recycle, consistently with a model of ligand-receptor reaction not limited by membrane diffusion. The use of N-cadherin-GFP receptors truncated or mutated in specific cytoplasmic regions show that N-cadherin turnover is exquisitely regulated by catenin partners. Turnover rates are considerably lower than those obtained previously in single molecule studies, demonstrating an active regulation of cadherin bond kinetics in intact cells. Finally, spontaneous neuronal contacts enriched in N-cadherin exhibited similar turnover rates, suggesting that such dynamics of N-cadherin may represent an intrinsic mechanism underlying the plasticity of neuronal adhesions.