Enzyme Conformational Transition Research Articles

Effects of Sodium Dodecyl Sulfate on the Enzyme Catalysis and Conformation of a Recombinant γ-Glutamyltranspeptidase from Bacillus licheniformis.

The study of interactions between proteins and surfactants is of relevance in a diverse range of applications including food, enzymatic detergent formulation, and drug delivery. In spite of sodium dodecyl sulfate(SDS)-induced unfolding has been studied in detail at the protein level, deciphering the conformation-activity relationship of a recombinant γ-glutamyltranspeptidase (BlrGGT) from Bacillus licheniformis remains important to understand how the transpeptidase activity is related to its conformation. In this study, we examined the enzyme catalysis and conformational transition of BlrGGT in the presence of SDS. Enzymatic assays showed that the transpeptidase activity of BlrGGT was greatly affected by SDS in a concentration-dependent manner with approximately 90% inactivation at 6mM. Native polyacrylamide gel electrophoresis of SDS-treated samples clearly revealed that the heterodimeric enzyme was apparently dissociated into two different subunits at concentrations above 2mM. The study of enzyme kinetics showed that SDS can act as a mixed-type inhibitor to reduce the catalytic efficiency of BlrGGT. Moreover, the t1/2 value of the enzyme at 55°C was greatly reduced from 495.1min to 7.4min in the presence of 1mM SDS. The I3/I1 ratio ofpyreneexcimerfluorescence emission changed around 3.7mMSDSin the absence ofBlrGGT and the inflection point of enzyme samples was reduced to less than 2.7mM. The Far-UV CD spectrum of the native enzyme had two negative peaks at 208 and 222nm, respectively; however, both negative peaks increased in magnitude with increasing SDS concentration and reached maximal values at above 4.0mM. Theintrinsic fluorescence spectraof tryptophan further demonstrated that the SDS-induced enzyme conformational transition occurred at approximately 5.1mM. Tween 20 significantly suppressed the interaction of BlrGGT with SDS by forming mixed micelles at a molar ratio of 1.0. Taken together, this study definitely promotes our better understanding of the relationship between the conformation and catalysis of BlrGGT.

Common Kinetic Mechanism of Abasic Site Recognition by Structurally Different Apurinic/Apyrimidinic Endonucleases.

Apurinic/apyrimidinic (AP) endonucleases Nfo (Escherichia coli) and APE1 (human) represent two conserved structural families of enzymes that cleave AP-site–containing DNA in base excision repair. Nfo and APE1 have completely different structures of the DNA-binding site, catalytically active amino acid residues and catalytic metal ions. Nonetheless, both enzymes induce DNA bending, AP-site backbone eversion into the active-site pocket and extrusion of the nucleotide located opposite the damage. All these stages may depend on local stability of the DNA duplex near the lesion. Here, we analysed effects of natural nucleotides located opposite a lesion on catalytic-complex formation stages and DNA cleavage efficacy. Several model DNA substrates that contain an AP-site analogue [F-site, i.e., (2R,3S)-2-(hydroxymethyl)-3-hydroxytetrahydrofuran] opposite G, A, T or C were used to monitor real-time conformational changes of the tested enzymes during interaction with DNA using changes in the enzymes’ intrinsic fluorescence intensity mainly caused by Trp fluorescence. The extrusion of the nucleotide located opposite F-site was recorded via fluorescence intensity changes of two base analogues. The catalytic rate constant slightly depended on the opposite-nucleotide nature. Thus, structurally different AP endonucleases Nfo and APE1 utilise a common strategy of damage recognition controlled by enzyme conformational transitions after initial DNA binding.

A Dynamical Model for Insulin Degrading Enzyme Conformational Transition between Closed and Open States

Insulin Degrading Enzyme (IDE) is involved in the deconstruction of insulin, amylin, and glucagon peptides involved in controlling blood glucose level and has also been found to break down amyloid-β, a peptide involved in Alzheimer's disease. IDE has two homologous N- and C-terminal domains, IDE-N and IDE-C. For IDE to degrade its substrates, both domains must come together for the catalytic cleft to assume closed conformation. New Cryo-Electron Microscopy (Cryo-EM) data suggests IDE passes through five conformations: completely closed with substrate bound, partially closed with substrate bound, partially open with substrate unbound, and open without substrate. Dynamical details of how these conformations connect are unknown. We investigated this process using long all atom molecular dynamics simulations (MD) of IDE monomer and dimer structures with and without substrate. Our simulations showed that IDE without insulin (pdb-code 4IOF) has larger relative motion between four subdomains (D1, D2, D3, D4) in the N- and C-terminal domains compared to an IDE with insulin (pdb-code 2WBY). Analysis of the insulin inside the catalytic chamber suggested that insulin was stabilized by interactions of its residues Gly1, Tyr14, Glu17 form its chain A and Phe1 and Ph25 from its chain B with IDE residues via a combination of hydrogen bonding and π - π interactions. We used, Essential Dynamics Analysis (EDA) to calculate principal normal modes of IDE motion from our trajectories. These normal modes were then applied as coordinate transformations combined with short MD simulations to gradually open the IDE-closed conformation. We used cross-comparison with Cryo-EM and hydrogen-deuterium exchange data of IDE as a guide to obtain a realistic model of how IDE changes between closed and open conformations.

Molecular simulations and free-energy calculations suggest conformation-dependent anion binding to a cytoplasmic site as a mechanism for Na+/K+-ATPase ion selectivity

Na+/K+-ATPase transports Na+ and K+ ions across the cell membrane via an ion-binding site becoming alternatively accessible to the intra- and extracellular milieu by conformational transitions that confer marked changes in ion-binding stoichiometry and selectivity. To probe the mechanism of these changes, we used molecular simulation and free-energy perturbation approaches to identify probable protonation states of Na+- and K+-coordinating residues in E1P and E2P conformations of Na+/K+-ATPase. Analysis of these simulations revealed a molecular mechanism responsible for the change in protonation state: the conformation-dependent binding of an anion (a chloride ion in our simulations) to a previously unrecognized cytoplasmic site in the loop between transmembrane helices 8 and 9, which influences the electrostatic potential of the crucial Na+-coordinating residue Asp926 This mechanistic model is consistent with experimental observations and provides a molecular-level picture of how E1P to E2P enzyme conformational transitions are coupled to changes in ion-binding stoichiometry and selectivity.

Mapping the Hydrogen Bond Networks in the Catalytic Subunit of Protein Kinase A Using H/D Fractionation Factors.

Protein kinase A is a prototypical phosphoryl transferase, sharing its catalytic core (PKA-C) with the entire kinase family. PKA-C substrate recognition, active site organization, and product release depend on the enzyme’s conformational transitions from the open to the closed state, which regulate its allosteric cooperativity. Here, we used equilibrium nuclear magnetic resonance hydrogen/deuterium (H/D) fractionation factors (φ) to probe the changes in the strength of hydrogen bonds within the kinase upon binding the nucleotide and a pseudosubstrate peptide (PKI5–24). We found that the φ values decrease upon binding both ligands, suggesting that the overall hydrogen bond networks in both the small and large lobes of PKA-C become stronger. However, we observed several important exceptions, with residues displaying higher φ values upon ligand binding. Notably, the changes in φ values are not localized near the ligand binding pockets; rather, they are radiated throughout the entire enzyme. We conclude that, upon ligand and pseudosubstrate binding, the hydrogen bond networks undergo extensive reorganization, revealing that the open-to-closed transitions require global rearrangements of the internal forces that stabilize the enzyme’s fold.

Critical role of γ-phosphate in structural transition of Na,K-ATPase upon ATP binding.

Active transport of sodium and potassium ions by Na,K-ATPase is accompanied by the enzyme conformational transition between E1 and E2 states. ATP and ADP bind to Na,K-ATPase in the E1 conformation with similar affinity but the properties of enzyme in complexes with these nucleotides are different. We have studied thermodynamics of Na,K-ATPase binding with adenine nucleotides at different temperatures using isothermal titration calorimetry. Our data indicate that β-phosphate is involved in complex formation by increasing the affinity of adenine nucleotides to Na,K-ATPase by an order of magnitude, while γ-phosphate does not affect it. ATP binding to Na,K-ATPase in contrast to ADP binding generates a structural transition in the enzyme, which is consistent with the movement of a significant portion of the surface area to a solvent-protected state. We propose that ATP binding leads to convergence of the nucleotide-binding and phosphorylation domains transferring the enzyme from the “E1-open” to “E1-closed” conformation ready for phosphorylation.

Kinetic characterization of the critical step in HIV-1 protease maturation

HIV maturation requires multiple cleavage of long polyprotein chains into functional proteins that include the viral protease itself. Initial cleavage by the protease dimer occurs from within these precursors, and yet only a single protease monomer is embedded in each polyprotein chain. Self-activation has been proposed to start from a partially dimerized protease formed from monomers of different chains binding its own N termini by self-association to the active site, but a complete structural understanding of this critical step in HIV maturation is missing. Here, we captured the critical self-association of immature HIV-1 protease to its extended amino-terminal recognition motif using large-scale molecular dynamics simulations, thus confirming the postulated intramolecular mechanism in atomic detail. We show that self-association to a catalytically viable state requires structural cooperativity of the flexible β-hairpin "flap" regions of the enzyme and that the major transition pathway is first via self-association in the semiopen/open enzyme states, followed by enzyme conformational transition into a catalytically viable closed state. Furthermore, partial N-terminal threading can play a role in self-association, whereas wide opening of the flaps in concert with self-association is not observed. We estimate the association rate constant (k(on)) to be on the order of ∼1 × 10(4) s(-1), suggesting that N-terminal self-association is not the rate-limiting step in the process. The shown mechanism also provides an interesting example of molecular conformational transitions along the association pathway.

Specific Sites in the Cytoplasmic N Terminus Modulate Conformational Transitions of the Na,K-ATPase

The cytoplasmic N terminus of the Na,K-ATPase is a highly charged and flexible structure that comprises three predicted helical regions including H1 spanning residues 27 to 33 and H2 spanning residues 42 to 50. Previous deletion mutagenesis experiments showed that deletion of residues up to and including most of H2 shifts the E(1)/E(2) conformational equilibrium toward E(1). The present study describes a clustered charge-to-alanine mutagenesis approach designed to delineate specific sites within the N terminus that modulate the steady-state E(1) <--> E(2) and E(1)P <--> E(2)P poise. Criteria to assess shifts in poise include (i) sensitivity to inhibition by inorganic orthovanadate to assess overall poise; (ii) K(+)-sensitivity of Na-ATPase measured at micromolar ATP to assess changes in the E(2)(K) + ATP --> E(1) x ATP + K(+) rate; (iii) K'(ATP) for low-affinity ATP binding at the latter step; (iv) overall catalytic turnover, and (v) the E(1)P --> E(2)P transition. The results of alanine replacements in H1 (31KKE) suggest that this site stabilizes E(2)P and to a lesser extent E(2). In H2, residues within 47HRK have a role in stabilizing E(2) but not E(2)P as revealed with double mutants 31KKE --> AAA/47H --> A and 31KKE --> AAA/47HRK --> AAA. Taken together, these observations suggest that sites 31KKE in H1 and 47HRK in H2 have distinct roles in modulating the enzyme's conformational transitions during the catalytic cycle of the enzyme.

Effect of ADP on Na +-Na + Exchange Reaction Kinetics of Na,K-ATPase

The whole-cell voltage-clamp technique was used in rat cardiac myocytes to investigate the kinetics of ADP binding to phosphorylated states of Na,K-ATPase and its effects on presteady-state Na +-dependent charge movements by this enzyme. Ouabain-sensitive transient currents generated by Na,K-ATPase functioning in electroneutral Na +-Na + exchange mode were measured at 23°C with pipette ADP concentrations ([ADP]) of up to 4.3 mM and extracellular Na + concentrations ([Na] o) between 36 and 145 mM at membrane potentials ( V M) from −160 to +80 mV. Analysis of charge- V M curves showed that the midpoint potential of charge distribution was shifted toward more positive V M both by increasing [ADP] at constant Na + o and by increasing [Na] o at constant ADP. The total quantity of mobile charge, on the other hand, was found to be independent of changes in [ADP] or [Na] o. The presence of ADP increased the apparent rate constant for current relaxation at hyperpolarizing V M but decreased it at depolarizing V M as compared to control (no added ADP), an indication that ADP binding facilitates backward reaction steps during Na +-Na + exchange while slowing forward reactions. Data analysis using a pseudo three-state model yielded an apparent K d of ∼6 mM for ADP binding to and release from the Na,K-ATPase phosphoenzyme; a value of 130 s −1 for k 2, a rate constant that groups Na + deocclusion/release and the enzyme conformational transition E 1∼P → E 2-P; a value of 162 s −1M −1 for k −2, a lumped second-order V M-independent rate constant describing the reverse reactions; and a Hill coefficient of ∼1 for Na + o binding to E 2-P. The results are consistent with electroneutral release of ADP before Na + is deoccluded and released through an ion well. The same approach can be used to study additional charge-moving reactions and associated electrically silent steps of the Na,K-pump and other transporters.

Complement activation in factor D-deficient mice.

To assess the contribution of the alternative pathway in complement activation and host defense and its possible role in the regulation of systemic energy balance in vivo, factor D-deficient mice were generated by gene targeting. The mutant mice have no apparent abnormality in development and their body weights are similar to those of factor D-sufficient littermates. Complement activation could not be initiated in the serum of deficient mice by the alternative pathway activators rabbit erythrocytes and zymosan. Surprisingly, injection of cobra venom factor (CVF) caused a profound and reproducible reduction in serum C3 levels, whereas, as expected, there was no C3 reduction in factor B-deficient mice treated similarly. Studies of C3 and factor B activation in vitro by CVF demonstrated that in factor D-deficient serum the alpha chain of C3 was cleaved gradually over a period of 60 min without detectable cleavage of factor B. CVF-dependent C3 cleavage in the deficient serum required the presence of Mg(2+), whereas in normal mouse serum the presence of divalent cations was not required. These results suggest that in mouse proteolytic cleavage of factor B by factor D is not an absolute requirement for the zymogen to active enzyme conformational transition of CVF-bound factor B. Kinetics of opsonization of Streptococcus pneumoniae by C3 fragments was much slower in factor D-deficient serum, suggesting a significant contribution of the alternative pathway to antibacterial host defense early after infection.

The magnitude of the allosteric conformational transition of aspartate transcarbamylase is altered by mutations.

Global conformational transitions are of central functional importance for many enzymes and binding proteins. It is not known, however, how much variability can exist in such structural-functional linkages. We have characterized the global magnitude of the T to R conformational transition of Escherichia coli aspartate transcarbamylase (ATCase) by measuring (1) hydration changes using osmotic stress and (2) hydrodynamic changes using high-precision analytical gel chromatography. We find that specific mutations can alter the structural magnitude of the enzyme's conformational transition without abolishing allostery, suggesting that some degree of plasticity exists in the conformational component of allostery.

The conformations of a functional spin-labeled derivative of gastric H/K-ATPase investigated by EPR spectroscopy

A spin-labeled derivative of porcine gastric H/K-ATPase with high ATP hydrolyzing activity (77 mumol of Pi/(mg.h)) has been prepared. Over 65% of initial ATPase activity (115 mumol of Pi/(mg.h)) was preserved after complete reaction of the enzyme with the lysine reactive nitroxide spin-labeled TEMPO isothiocyanate (TITC). In contrast, rapid and complete loss of ATPase activity occurred after reaction of the enzyme with the lysine directed fluorescent probe FITC. Conventional EPR spectra of TITC labeled H/K-ATPase reflected mainly the slow rotational diffusion of the enzyme in the membrane. An upper limit enzyme intramembranous radius of 108 A was calculated on the basis of rotational correlation times estimated from saturation transfer (ST) EPR spectral lineshapes. Conventional EPR spectra exhibited two major components corresponding to at least two populations of strongly constrained spin-labels. Difference spectroscopy revealed that the proportion of these two components changed markedly with temperature. Moreover, the proportion of the components was sensitive to the presence of the activating ionic ligands Mg2+ and ATP, which induce enzyme conformational transitions, and to the reversible inhibitor SCH 28080, which binds to the K+ sensitive form of the enzyme. These findings show that EPR spectroscopy is able to report functionally coupled conformational changes of gastric H/K-ATPase and imply that the spin-labels are attached to lysines within functionally important regions of the enzyme.

Modification of lysine 501 in Na,K-ATPase reveals coupling between cation occupancy and changes in the ATP binding domain

Treatment of the canine renal Na,K-ATPase with N-(2-nitro-4-isothiocyanophenyl)-imidazole (NIPI) results in irreversible loss of enzymatic activity. The reactivity of the protein toward the probe is about 10-fold greater in the E1.Na or sodium-bound form than when it is in the E2.K or potassium-bound form. Fully inactivated enzyme does not bind ATP but binds sodium and potassium and undergoes the major enzyme conformational transitions (Ellis-Davies, G. C. R., and Kaplan, J. H. (1990) J. Biol. Chem. 265, 20570-20576). Labeling of the sodium pump protein with [3H]NIPI in either the E1.Na or E2.K conformations results in the covalent incorporation of one molecule of probe per alpha-subunit of ATPase. Trypsin treatment of purified alpha-subunit, and separation of the digest using reverse-phase high performance liquid chromatography, yields five major radioactive fragments in each case. Amino acid sequencing indicates that only one residue, lysine 501, is labeled by NIPI. This suggests that the integrity of the domain containing Lys501 is essential for both high and low affinity binding of ATP by the Na,K-ATPase. Furthermore, the spatial organization of the protein close to lysine 501 is changed in the E1.Na and E2.K form of the enzyme. This change is reflected in the greater reactivity of lysine 501 in the E1.Na conformation and indicates that the binding of either sodium or potassium ions to their sites on the sodium pump has differential effects on the nucleotide binding domain containing lysine 501.

Participation of hydrogen ion in the calcium-induced conformational transition of 4-nitro-2,1,3-benzoxadiazole-labeled sarcoplasmic reticulum ATPase

The binding of Ca2+ to 4-nitro-2,1,3-benzoxadiazole (NBD)-labeled sarcoplasmic reticulum Ca2(+)-ATPase was accelerated markedly when the pH was changed at 11 degrees C from 6.5 to 8.0 at the time of Ca2+ addition. We examined the effect of pH on the enzyme conformational transition by measuring the kinetics of NBD fluorescence rises induced by a pH jump under various ligand conditions. The fast fluorescence rise following a pH jump from 6.0 or 6.5 to various test pHs in the presence and absence of Ca2+ proceeded monoexponentially. The amplitude of this fluorescence rise in the presence of Ca2+ was independent of the test pH, whereas the observed rate constant (kobs) increased markedly as the test pH increased. In contrast, the amplitude of the fast fluorescence rise in the absence of Ca2+ increased with increasing test pH, whereas kobs decreased. MgATP or Mg2+ influenced the pH dependences of these parameters in a complex way except for the amplitudes measured in the presence of Ca2+. These data could be simulated by using a reaction model in which Ca2+ binding is preceded by a rate-limiting enzyme conformational transition from a low to a high NBD fluorescence state and 1 mol each of H+ is liberated before and after this conformational transition. MgATP or Mg2+ appeared to promote this conformational transition by enhancing deprotonation of the enzyme. These results suggest that deprotonation may be the primary event in the activation of the unphosphorylated enzyme by Ca2+.

The influence of cytoplasmic sodium concentration on the stoichiometry of the sodium pump.

Using inside-out vesicles of human red cell membranes, the effects of cytoplasmic Na+ in the range 0-5 mM on ATP-dependent 22Na+ influx (normal efflux) and 86Rb+ efflux (normal influx) were tested. The sodium pump stoichiometry, i.e. the ratio of net 22Na+ influx:86Rb+ efflux was reduced markedly when the cytoplasmic Na+ was reduced to less than 1 mM. Reduction in cytoplasmic Na+ concentration was associated also with a decreased sensitivity of the pump to effects of extracellular Rb+. Thus, extracellular (intravesicular) Rb+ stimulation observed at high ATP concentration and inhibition observed at low ATP concentration were not observed when the cytoplasmic (extravesicular) Na+ concentration was reduced to less than or equal to 0.2 mM. It is suggested that at low cytoplasmic Na+, the pump can operate with less than maximal sites filled with Na+ ions. Under this condition, it is likely that an enzymic step associated with either the ion translocation step or the enzyme's conformational transition becomes rate-limiting.

Self-organization and dynamics of an open futile cycle

The dynamic behaviour of an open futile cycle composed of two enzymes has been investigated in the vicinity of a steady-state. A necessary condition required for damped or sustained oscillations of the system is that enzyme E 2, which controls recycling of the substrate S 2, be inhibited by an excess of this substrate. In order for the system to be neutrally stable and therefore to exhibit sustained oscillations, it is not necessary for antagonist enzyme E 1 to be activated by its product S 2. If it is enzyme E 1 which is inhibited by an excess of its substrate S 1, the system has a saddle point. Other conditions for stability or instability of the system have been determined. If the enzyme E 1, which is not inhibited by the substrate, exhibits a slow conformational transition of the mnemonical type, this transition dramatically alters the stability behavior of the system. If the mnemonical enzyme E 1 were exhibiting a positive kinetic co-operativity, decreasing the rate of the conformational transition of the mnemonical enzyme will increase the stability of the whole system and will tend to damp the oscillations in the vicinity of the steady-state. If conversely the mnemonical enzyme E 1 were exhibiting a negative kinetic co-operativity, decreasing the rate of the enzyme conformational transition will decrease the stability of the system and will tend to create or amplify oscillations of the system taken as a whole. If these results may be extended to more complex metabolic cycles, involving more than two enzymes, it may be tentatively considered that positive co-operativity associated with slow transition has emerged in the course of evolution in order to limit temporal instabilities of metabolic cycles. Alternatively one may speculate that the “biological function” of negative co-operativity is to create or amplify these temporal instabilities.