The role of slow and fast protein motions in allosteric interactions

Allostery is fundamentally thermodynamic in nature. Long-range communication in proteins may be mediated not only by changes in the mean conformation with enthalpic contribution but also by changes in dynamic fluctuations with entropic contribution. The important role of protein motions in mediating allosteric interactions has been established by NMR spectroscopy. By using CAP as a model system, we have shown how changes in protein structure and internal dynamics can allosterically regulate protein function and activity. The results indicate that changes in conformational entropy can give rise to binding enhancement, binding inhibition, or have no effect in the expected affinity, depending on the magnitude and sign of enthalpy–entropy compensation. Moreover, allosteric interactions can be regulated by the modulation a low-populated conformation states that serve as on-pathway intermediates for ligand binding. Taken together, the interplay between fast internal motions, which are intimately related to conformational entropy, and slow internal motions, which are related to poorly populated conformational states, can regulate protein activity in a way that cannot be predicted on the basis of the protein’s ground-state structure.     

Protein dynamics from NMR

This review surveys recent investigations of conformational fluctuations of proteins in solution using NMR techniques. Advances in experimental methods have provided more accurate means of characterizing fast and slow internal motions as well as overall diffusion. The information obtained from NMR dynamics experiments provides insights into specific structural changes or configurational energetics associated with function. A variety of applications illustrate that studies of protein dynamics provide insights into protein-protein interactions, target recognition, ligand binding, and enzyme function.     

Guanine nucleotides differentially modulate backbone dynamics of the STAS domain of the SulP/SLC26 transport protein Rv1739c of Mycobacterium tuberculosis

Enzymatic catalysis and protein signaling are dynamic processes that involve local and/or global conformational changes occurring across a broad range of time scales. (1) H-(15) N relaxation NMR provides a comprehensive understanding of protein backbone dynamics both in the apo (unliganded) and ligand-bound conformations, enabling both fast and slow internal motions of individual amino acid residues to be observed. We recently reported the structure and nucleotide binding properties of the sulfate transporter and anti-sigma factor antagonist (STAS) domain of Rv1739c, a SulP anion transporter protein of Mycobacterium tuberculosis. In the present study, we report (1) H-(15) N NMR backbone dynamics measurements [longitudinal (T(1) ), transverse (T(2) ) and steady-state ({(1) H}-(15) N) heteronuclear NOE] of the Rv1739c STAS domain, in the absence and presence of saturating concentrations of GTP and GDP. Analysis of measured relaxation data and estimated dynamic parameters indicated distinct features differentiating the binding of GTP and GDP to Rv1739c STAS. The 9.55 ns overall rotational correlation time of Rv1739c STAS increased to 10.48 ns in the presence of GTP, and to 13.25 ns in the presence of GDP, indicating significant nucleotide-induced conformational changes. These conformational changes were accompanied by slow time scale (μs to ms) motions in discrete regions of the protein, as reflected by guanine nucleotide-induced changes in relaxation parameters. The observed nucleotide-specific alterations in the relaxation properties of individual STAS residues reflect an increased molecular anisotropy and/or the emergence of conformational equilibria governing functional properties of the STAS domain.     

Geometry-based sampling of conformational transitions in proteins

The fast and accurate prediction of protein flexibility is one of the major challenges in protein science. Enzyme activity, signal transduction, and ligand binding are dynamic processes involving essential conformational changes ranging from small side chain fluctuations to reorientations of entire domains. In the present work, we describe a reimplementation of the CONCOORD approach, termed tCONCOORD, which allows a computationally efficient sampling of conformational transitions of a protein based on geometrical considerations. Moreover, it allows for the extraction of the essential degrees of freedom, which, in general, are the biologically relevant ones. The method rests on a reliable estimate of the stability of interactions observed in a starting structure, in particular those interactions that change during a conformational transition. Applications to adenylate kinase, calmodulin, aldose reductase, T4-lysozyme, staphylococcal nuclease, and ubiquitin show that experimentally known conformational transitions are faithfully predicted.     

THz time scale structural rearrangements and binding modes in lysozyme-ligand interactions

Predicting the conformational changes in proteins that are relevant for substrate binding is an ongoing challenge in the aim of elucidating the functional states of proteins. The motions that are induced by protein-ligand interactions are governed by the protein global modes. Our measurements indicate that the detected changes in the global backbone motion of the enzyme upon binding reflect a shift from the large-scale collective dominant mode in the unbound state towards a functional twisting deformation that assists in closing the binding cleft. Correlated motion in lysozyme has been implicated in enzyme function in previous studies, but detailed characterization of the internal fluctuations that enable the protein to explore the ensemble of conformations that ultimately foster large-scale conformational change is yet unknown. For this reason, we use THz spectroscopy to investigate the picosecond time scale binding modes and collective structural rearrangements that take place in hen egg white lysozyme (HEWL) when bound by the inhibitor (NAG) ₃. These protein thermal motions correspond to fluctuations that have a role in both selecting and sampling from the available protein intrinsic conformations that communicate function. Hence, investigation of these fast, collective modes may provide knowledge about the mechanism leading to the preferred binding process in HEWL-(NAG) ₃. Specifically, in this work we find that the picosecond time scale hydrogen-bonding rearrangements taking place in the protein hydration shell with binding modify the packing density within the hydrophobic core on a local level. These localized, intramolecular contact variations within the protein core appear to facilitate the large cooperative movements within the interfacial region separating the α- and β- domain that mediate binding. The THz time-scale fluctuations identified in the protein-ligand system may also reveal a molecular mechanism for substrate recognition.     

Conformational transitions in protein-protein association: binding of fasciculin-2 to acetylcholinesterase

The neurotoxin fasciculin-2 (FAS2) is a picomolar inhibitor of synaptic acetylcholinesterase (AChE). The dynamics of binding between FAS2 and AChE is influenced by conformational fluctuations both before and after protein encounter. Submicrosecond molecular dynamics trajectories of apo forms of fasciculin, corresponding to different conformational substates, are reported here with reference to the conformational changes of loop I of this three-fingered toxin. This highly flexible loop exhibits an ensemble of conformations within each substate corresponding to its functions. The high energy barrier found between the two major substates leads to transitions that are slow on the timescale of the diffusional encounter of noninteracting FAS2 and AChE. The more stable of the two apo substates may not be the one observed in the complex with AChE. It seems likely that the more stable apo form binds rapidly to AChE and conformational readjustments then occur in the resulting encounter complex.     

A Physical Picture of Protein Dynamics and Conformational Changes

A physical model is reviewed which explains different aspects of protein dynamics consistently. At low temperatures, the molecules are frozen in conformational substates. Their average energy is 3/2RT. Solid-state vibrations occur on a time scale of femtoseconds to nanoseconds. Above a characteristic temperature, often called the dynamical transition temperature, slow modes of motions can be observed occurring on a time scale between about 140 and 1 ns. These motions are overdamped, quasidiffusive, and involve collective motions of segments of the size of an α-helix. Molecules performing these types of motion are in the “flexible state”. This state is reached by thermal activation. It is shown that these motions are essential for conformational relaxation. Based on this picture, a new approach is proposed to understand conformational changes. It connects structural fluctuations and conformational transitions.     

Pre-steady-state kinetics shows differences in processing of various DNA lesions by Escherichia coli formamidopyrimidine-DNA glycosylase

Formamidopyrimidine-DNA-glycosylase (Fpg pro tein, MutM) catalyses excision of 8-oxoguanine (8-oxoG) and other oxidatively damaged purines from DNA in a glycosylase/apurinic/apyrimidinic-lyase reaction. We report pre-steady-state kinetic analysis of Fpg action on oligonucleotide duplexes containing 8-oxo-2′-deoxyguanosine, natural abasic site or tetrahydrofuran (an uncleavable abasic site analogue). Monitoring Fpg intrinsic tryptophan fluorescence in stopped-flow experiments reveals multiple conformational transitions in the protein molecule during the catalytic cycle. At least four and five conformational transitions occur in Fpg during the interaction with abasic and 8-oxoG-containing substrates, respectively, within 2 ms to 10 s time range. These transitions reflect the stages of enzyme binding to DNA and lesion recognition with the mutual adjustment of DNA and enzyme structures to achieve catalytically competent conformation. Unlike these well-defined binding steps, catalytic stages are not associated with discernible fluorescence events. Only a single conformational change is detected for the cleavable substrates at times exceeding 10 s. The data obtained provide evidence that several fast sequential conformational changes occur in Fpg after binding to its substrate, converting the protein into a catalytically active conformation.     

Multiple Discrete Transitions Underlie Voltage-Dependent Activation in CLC Cl/H Antiporters

Most mammalian chloride channels and transporters in the CLC family display pronounced voltage-dependent gating. Surprisingly, despite the complex nature of the gating process and the large contribution to it by the transport substrates, experimental investigations of the fast gating process usually produce canonical Boltzmann activation curves that correspond to a simple two-state activation. By using nonlinear capacitance measurements of two mutations in the ClC-5 transporter, here we are able to discriminate and visualize discrete transitions along the voltage-dependent activation pathway. The strong and specific dependence of these transitions on internal and external [Cl⁻] suggest that CLC gating involves voltage-dependent conformational changes as well as coordinated movement of transported substrates.     

Functional Roles of Slow Enzyme Conformational Changes in Network Dynamics

Extensive studies from different fields reveal that many macromolecules, especially enzymes, show slow transitions among different conformations. This phenomenon is named such things as dynamic disorder, heterogeneity, hysteretic or mnemonic enzymes across these different fields, and has been directly demonstrated by single molecule enzymology and NMR studies recently. We analyzed enzyme slow conformational changes in the context of regulatory networks. A single enzymatic reaction with slow conformational changes can filter upstream network noises, and can either resonantly respond to the system stimulus at certain frequencies or respond adaptively for sustained input signals of the network fluctuations. It thus can serve as a basic functional motif with properties that are normally for larger intermolecular networks in the field of systems biology. We further analyzed examples including enzymes functioning against pH fluctuations, metabolic state change of Artemia embryos, and kinetic insulation of fluctuations in metabolic networks. The study also suggests that hysteretic enzymes may be building blocks of synthetic networks with various properties such as narrow-banded filtering. The work fills the missing gap between studies on enzyme biophysics and network level dynamics, and reveals that the coupling between the two is functionally important; it also suggests that the conformational dynamics of some enzymes may be evolutionally selected.     

Side-chain conformational changes upon Protein-Protein Association

Conformational changes upon protein-protein association are the key element of the binding mechanism. The study presents a systematic large-scale analysis of such conformational changes in the side chains. The results indicate that short and long side chains have different propensities for the conformational changes. Long side chains with three or more dihedral angles are often subject to large conformational transition. Shorter residues with one or two dihedral angles typically undergo local conformational changes not leading to a conformational transition. A relationship between the local readjustments and the equilibrium fluctuations of a side chain around its unbound conformation is suggested. Most of the side chains undergo larger changes in the dihedral angle most distant from the backbone. The frequencies of the core-to-surface interface transitions of six nonpolar residues and Tyr are larger than the frequencies of the opposite surface-to-core transitions. The binding increases both polar and nonpolar interface areas. However, the increase of the nonpolar area is larger for all considered classes of protein complexes, suggesting that the protein association perturbs the unbound interfaces to increase the hydrophobic contribution to the binding free energy. To test modeling approaches to side-chain flexibility in protein docking, conformational changes in the X-ray set were compared with those in the docking decoy sets. The results lead to a better understanding of the conformational changes in proteins and suggest directions for efficient conformational sampling in docking protocols.     

The role of interchain disulphide bridges in the conformational stability of human immunoglobulin G1 subclass. Hydrogen-deuterium exchange studies.

The hydrogen-deuterium exchange data of human immunoglobulin G1 (IgG1) are interpreted by assuming fast fluctuations of the protein conformation, through which the peptide groups become exposed to the solvent. The probability of solvent exposure of peptide hydrogens reflects a rather loose conformation for native IgG in comparison with other globular proteins. The probability of solvent exposure is greater than 10(-3) for half of the peptide groups, which shows that the conformational transitions by which these groups are exposed to the solvent are accompanied by changes in standard free energy less than 17 kJ/mol (4 kcal/mol). In the range of pH 6.2-8.45, at 25 degrees C no gross conformational changes are reflected in the hydrogen-deuterium exchange behaviour of the native, the reduced-nonalkylated-reassociated and the reduced-S-alkylated-reassociated IgG1. No difference could be detected in the conformational stability of the native and reoxidised reassociated IgG1 proteins. The lack of inter-subunit disulphide bridges in S-alkylated-reassociated molecules results in an increased conformational motility. This destabilization of protein conformation affects about 90% of the peptide groups covered by the measurements, and corresponds to changes in standard free energy of 8 kJ/mol on the average.     

Conformational changes in the chaperonin GroEL: new insights into the allosteric mechanism

Conformational changes are known to play a crucial role in the function of the bacterial GroE chaperonin system. Here, results are presented from an essential dynamics analysis of known experimental structures and from computer simulations of GroEL using the CONCOORD method. The results indicate a possible direct form of inter-ring communication associated with internal fluctuations in the nucleotide-binding domains upon nucleotide and GroES binding that are involved in the allosteric mechanism of GroEL. At the level of conformational transitions in entire GroEL rings, nucleotide-induced structural changes were found to be distinct and in principle uncoupled from changes occurring upon GroES binding. However, a coupling is found between nucleotide-induced conformational changes and GroES-mediated transitions, but only in simulations of GroEL double rings, and not in simulations of single rings. This provides another explanation for the fact that GroEL functions a double ring system.     

Ligand-induced changes in the conformational stability and flexibility of glutamate dehydrogenase and their role in catalysis and regulation

Bovine glutamate dehydrogenase (GDH) is allosterically regulated and requires substrate‐induced subunit interactions for maximum catalytic activity. Steady‐state and presteady‐state kinetics indicate that the rate‐limiting step depends on the nature of the substrate and are likely associated with conformational fluctuations necessary for optimal hydride transfer. Deuterated glutamate shows a steady‐state isotope effect but no effect on the presteady‐state burst rate, demonstrating that conformational effects are rate limiting for hydride transfer while product release is overall rate limiting for glutamate. Guanidine hydrochloride unfolding, heat inactivation, and differential scanning calorimetry demonstrate the effects of alternative substrates, glutamate and norvaline, on conformational stability. Glutamate has little effect on overall stability, whereas norvaline markedly stabilizes the protein. Limited proteolysis demonstrates that glutamate had a variety of effects on local flexibility, whereas norvaline significantly decreased conformational fluctuations that allow protease cleavage. Dynamic light scattering suggests that norvaline stabilizes all interfaces in the hexamer, whereas glutamate had little effect on trimer–trimer interactions. The substrate glutamate exhibits negative cooperativity and complex allosteric regulation but has only minor effects on global GDH stability, while promoting certain local conformational fluctuations. In contrast, the substrate norvaline does not show negative cooperativity or allow allosteric regulation. Instead, norvaline significantly stabilizes the enzyme and markedly slows or prevents local conformational fluctuations that are likely to be important for cooperative effects and to determine the overall rate of hydride transfer. This suggests that homotropic allosteric regulation by the enzymatic substrate involves changes in both global stability and local flexibility of the protein.     

Substrate-induced exposure of an energy-coupling motif of a membrane transporter

BtuB is an outer membrane protein responsible for the uptake of vitamin B12 by Escherichia coli. It belongs to a family of bacterial transport proteins that derive energy for transport by coupling to the trans-periplasmic energy-coupling protein TonB. Using site-directed spin labeling and EPR we investigated the structure and substrate-induced changes in the TonB box, a highly conserved region in all TonB dependent transporters that may couple to TonB. In the absence of substrate, the line widths and collision parameters from EPR are consistent with this domain existing in a structured helical conformation that contacts the barrel of the transporter. Addition of substrate converts this segment into an extended structure that is highly dynamic, disordered and probably extended into the periplasm. This structural change demonstrates that the TonB box cycles between sequestered and accessible states in a substrate-dependent fashion. In a transport defective mutant of BtuB, this conformational cycle is disrupted and the TonB box appears to be extended even in the absence of substrate. These data suggest that the TonB box extends into the periplasm and interacts with TonB only in     

Solution NMR Studies of Chlorella Virus DNA Ligase-adenylate

DNA ligases are essential guardians of genome integrity by virtue of their ability to recognize and seal 3′-OH/5′-phosphate nicks in duplex DNA. The substrate binding and three chemical steps of the ligation pathway are coupled to global and local changes in ligase structure, involving both massive protein domain movements and subtle remodeling of atomic contacts in the active site. Here we applied solution NMR spectroscopy to study the conformational dynamics of the Chlorella virus DNA ligase (ChVLig), a minimized eukaryal ATP-dependent ligase consisting of nucleotidyltransferase, OB, and latch domains. Our analysis of backbone ¹⁵N spin relaxation and ¹⁵N,¹H residual dipolar couplings of the covalent ChVLig-AMP intermediate revealed conformational sampling on fast (picosecond to nanosecond) and slow timescales (microsecond to millisecond), indicative of interdomain and intradomain flexibility. We identified local and global changes in ChVLig-AMP structure and dynamics induced by phosphate. In particular, the chemical shift perturbations elicited by phosphate were clustered in the peptide motifs that comprise the active site. We hypothesize that phosphate anion mimics some of the conformational transitions that occur when ligase-adenylate interacts with the nick 5′-phosphate.     

Flap opening and dimer-interface flexibility in the free and inhibitor-bound HIV protease, and their implications for function.

BACKGROUND: (1)H and (15)N transverse relaxation measurements on perdeuterated proteins are ideally suited for detecting backbone conformational fluctuations on the millisecond-microsecond timescale. The identification of conformational exchange on this timescale by measuring the relaxation of both (1)H and (15)N holds great promise for the elucidation of functionally relevant conformational changes in proteins. RESULTS: We measured the transverse (1)H and (15)N relaxation rates of backbone amides of HIV-1 protease in its free and inhibitor-bound forms. An analysis of these rates, obtained as a function of the effective rotating frame field, provided information about the timescale of structural fluctuations in several regions of the protein. The flaps that cover the active site of the inhibitor-bound protein undergo significant changes of backbone (φ,psi) angles, on the 100 micros timescale, in the free protein. In addition, the intermonomer beta-sheet interface of the bound form, which from protease structure studies appears to be rigid, was found to fluctuate on the millisecond timescale. CONCLUSIONS: We present a working model of the flap-opening mechanism in free HIV-1 protease which involves a transition from a semi-open to an open conformation that is facilitated by interaction of the Phe53 ring with the substrate. We also identify a surprising fluctuation of the beta-sheet intermonomer interface that suggests a structural requirement for maturation of the protease. Thus, slow conformational fluctuations identified by (1)H and (15)N transverse relaxation measurements can be related to the biological functions of proteins.     

Role of induced fit in enzyme specificity: a molecular forward/reverse switch

Enzyme structures solved with and without bound substrate often show that substrate-induced conformational changes bring catalytic residues into alignment, alter the local environment, and position the substrate for catalysis. Although the structural data are compelling, the role of conformational changes in enzyme specificity has been controversial in that specificity is a kinetic property that is not easy to predict based upon structure alone. Recent studies on DNA polymerization have illuminated the role of substrate-induced conformational changes in enzyme specificity by showing that the rate at which the enzyme opens to release the bound substrate is a key kinetic parameter. The slow release of a correct substrate commits it to the forward reaction so that specificity is determined solely by the rate of substrate binding, including the isomerization step, and not by the slower rate of the chemical reaction. In contrast, fast dissociation of an incorrect substrate favors release rather than reaction. Thus, the conformational change acts as a molecular switch to select the right substrate and to recognize and disfavor the reaction of an incorrect substrate. A conformational switch may also favor release rather than reverse reaction of the product.     

Molecular basis for pH sensitivity and proton transfer in green fluorescent protein: protonation and conformational substates from electrostatic calculations.

We performed a theoretical study to elucidate the coupling between protonation states and orientation of protein dipoles and buried water molecules in green fluorescent protein, a versatile biosensor for protein targeting. It is shown that the ionization equilibria of the wild-type green fluorescent protein-fluorophore and the internal proton-binding site E222 are mutually interdependent. Two acid-base transitions of the fluorophore occur in the presence of neutral (physiologic pH) and ionized (pH > 12) E222, respectively. In the pH-range from approximately 8 to approximately 11 ionized and neutral sites are present in constant ratio, linked by internal proton transfer. The results indicate that modulation of the internal proton sharing by structural fluctuations or chemical variations of aligning residues T203 and S65 cause drastic changes of the neutral/anionic ratio-despite similar physiologic fluorophore pK(a) s. Moreover, we find that dipolar heterogeneities in the internal hydrogen-bond network lead to distributed driving forces for excited-state proton transfer. A molecular model for the unrelaxed surrounding after deprotonation is discussed in relation to pathways providing fast ground-state recovery or slow stabilization of the anion. The calculated total free energy for excited-state deprotonation ( approximately 19 k(B)T) and ground-state reprotonation ( approximately 2 k(B)T) is in accordance with absorption and emission data (相似文献   

Probing the effect of transport inhibitors on the conformation of the mitochondrial citrate transport protein via a site-directed spin labeling approach

The present investigation utilized the site-directed spin labeling method of electron paramagnetic resonance (EPR) spectroscopy to identify the effect of citrate, the natural ligand, and transport inhibitors on the conformation of the yeast mitochondrial citrate transport protein (CTP) reconstituted in liposomal vesicles. Spin label was placed at six different locations within the CTP in order to monitor conformational changes that occurred near each of the transporter’s two substrate binding sites, as well as at more distant domains within the CTP architecture. We observed that citrate caused little change in the EPR spectra. In contrast the transport inhibitors 1,2,3-benzenetricarboxylate (BTC), pyridoxal 5′-phosphate (PLP), and compound 792949 resulted in spectral changes that indicated a decrease in the flexibility of the attached spin label at each of the six locations tested. The rank order of the immobilizing effect was compound 792949 > PLP > BTC. The four spin-label locations that report on the CTP substrate binding sites displayed the greatest changes in the EPR spectra upon addition of inhibitor. Furthermore, we found that when compound 792949 was added vectorially (i.e., extra- and/or intra-liposomally), the immobilizing effect was mediated nearly exclusively by external reagent. In contrast, upon addition of PLP vectorially, the effect was mediated to a similar extent from both the external and the internal compartments. In combination our data indicate that: i) citrate binding to the CTP substrate binding sites does not alter side-chain and/or backbone mobility in a global manner and is consistent with our expectation that both in the absence and presence of substrate the CTP displays the flexibility required of a membrane transporter; and ii) binding of each of the transport inhibitors tested locked multiple CTP domains into more rigid conformations, thereby exhibiting long-range inter-domain conformational communication. The differential vectorial effects of compound 792949 and PLP are discussed in the context of the CTP homology-modeled structure and potential mechanistic molecular explanations are given.