Dynamic allosteric communication pathway directing differential activation of the glucocorticoid receptor.

Allosteric communication within proteins is a hallmark of biochemical signaling, but the dynamic transmission pathways remain poorly characterized. We combined NMR spectroscopy and surface plasmon resonance to reveal these pathways and quantify their energetics in the glucocorticoid receptor, a transcriptional regulator controlling development, metabolism, and immune response. Our results delineate a dynamic communication network of residues linking the ligand-binding pocket to the activation function-2 interface, where helix 12, a switch for transcriptional activation, exhibits ligand- and coregulator-dependent dynamics coupled to graded activation. The allosteric free energy responds to variations in ligand structure: subtle changes gradually tune allostery while preserving the transmission pathway, whereas substitution of the entire pharmacophore leads to divergent allosteric control by apparently rewiring the communication network. Our results provide key insights that should aid in the design of mechanistically differentiated ligands.

Designing interactions by control of protein-ligand complex conformation: tuning arginine-arene interaction geometry for enhanced electrostatic protein-ligand interactions.

We investigated galectin-3 binding to 3-benzamido-2-O-sulfo-galactoside and -thiodigalactoside ligands using a combination of site-specific mutagenesis, X-ray crystallography, computational approaches, and binding thermodynamics measurements. The results reveal a conformational variability in a surface-exposed arginine (R144) side chain in response to different aromatic C3-substituents of bound galactoside-based ligands. Fluorinated C3-benzamido substituents induced a shift in the side-chain conformation of R144 to allow for an entropically favored electrostatic interaction between its guanidine group and the 2-O-sulfate of the ligand. By contrast, binding of ligands with non-fluorinated substituents did not trigger a conformational change of R144. Hence, a sulfate-arginine electrostatic interaction can be tuned by the choice of ligand C3-benzamido structures to favor specific interaction modes and geometries. These results have important general implications for ligand design, as the proper choice of arginine-aromatic interacting partners opens up for ligand-controlled protein conformation that in turn may be systematically exploited in ligand design.

Dynamics of the transition between open and closed conformations in a calmodulin C-terminal domain mutant.

BACKGROUND: Calmodulin is a ubiquitous Ca(2+)-activated regulator of cellular processes in eukaryotes. The structures of the Ca(2+)-free (apo) and Ca(2+)-loaded states of calmodulin have revealed that Ca(2+) binding is associated with a transition in each of the two domains from a closed to an open conformation that is central to target recognition. However, little is known about the dynamics of this conformational switch. RESULTS: The dynamics of the transition between closed and open conformations in the Ca(2+)-loaded state of the E140Q mutant of the calmodulin C-terminal domain were characterized under equilibrium conditions. The exchange time constants (tau(ex)) measured for 42 residues range from 13 to 46 micros, with a mean of 21 +/- 3 micros. The results suggest that tau(ex) varies significantly between different groups of residues and that residues with similar values exhibit spatial proximity in the structures of apo and/or Ca(2+)-saturated wild-type calmodulin. Using data for one of these groups, we obtained an open population of p(o) = 0.50 +/- 0.17 and a closed --> open rate constant of k(o) = x 10(4) s(-1). CONCLUSIONS: The conformational exchange dynamics appear to involve locally collective processes that depend on the structural topology. Comparisons with previous results indicate that similar processes occur in the wild-type protein. The measured rates match the estimated Ca(2+) off rate, suggesting that Ca(2+) release may be gated by the conformational dynamics. Structural interpretation of estimated chemical shifts suggests a mechanism for ion release.

Solution structure of (Cd2+)1-calbindin D9k reveals details of the stepwise structural changes along the Apo-->(Ca2+)II1-->(Ca2+)I,II2 binding pathway.

The three-dimensional solution structure of (Cd2+)1-calbindin D9k has been determined by distance geometry, restrained molecular dynamics and relaxation matrix calculations using experimental constraints obtained from two-dimensional 1H and 15N-1H NMR spectroscopy. The final input data consisted of 1055 NOE distance constraints and 71 dihedral angle constraints, corresponding to 15 constraints per residue on average. The resulting ensemble of 24 structures has no distance or dihedral angle constraints consistently violated by more than 0.07 A and 1.8 degrees, respectively. The structure is characteristic of an EF-hand protein, with two helix-loop-helix calcium binding motifs joined by a flexible linker, and a short anti-parallel beta-type interaction between the two ion-binding sites. The four helices are well defined with a root mean square deviation from the mean coordinates of 0.35 A for the backbone atoms. The structure of the half-saturated cadmium state was compared with the previously determined solution structures of the apo and fully calcium saturated calbindin D9k. The comparisons were aided by introducing the ensemble averaged distance difference matrix as a tool for analyzing differences between two ensembles of structures. Detailed analyses of differences between the three states in backbone and side-chain dihedral angles, hydrogen bonds, interatomic distances, and packing of the hydrophobic core reveal the reorganization of the protein that occurs upon ion binding. Overall, it was found that (Cd2+)1-calbindin D9k, representing the half-saturated calcium state with an ion in site II, is structurally more similar to the fully calcium-saturated state than the apo state. Thus, for the binding sequence apo-->(Ca2+)II1-->(Ca2+)I,II2, the structural changes occurring upon ion binding are most pronounced for the first binding step, an observation that bears significantly on the molecular basis for cooperative calcium binding in calbindin D9k.

Backbone dynamics of Escherichia coli ribonuclease HI: correlations with structure and function in an active enzyme.

Ribonuclease H is an endonuclease that hydrolyzes the RNA moiety of RNA-DNA duplex molecules. Escherichia coli ribonuclease H is involved in DNA replication, and retroviral ribonuclease H is essential for reverse transcription of the viral genome. To characterize the intramolecular dynamical properties of E. coli ribonuclease H, spin-lattice relaxation rate constants, spin-spin relaxation rate constants and steady state nuclear Overhauser effects for the 15N nuclear spins were measured by using proton-detected heteronuclear NMR spectroscopy. The relaxation data were analyzed by using a series of dynamical models in conjunction with a statistical model selection protocol. Ribonuclease H exhibits a complex array of dynamical features, most notably in the parallel beta-strands of the principal five-stranded beta-sheet, the coiled-coil helical interface, the active site, and the loop regions surrounding the active site. The dynamical properties are correlated with local structural environments of the 15N spins and suggest possible relationships to the functional properties of ribonuclease H. Results for E. coli ribonuclease H are compared to previously reported results for the human immunodeficiency virus type 1 ribonuclease H domain of reverse transcriptase.

Molecular basis for co-operativity in Ca2+ binding to calbindin D9k. 1H nuclear magnetic resonance studies of (Cd2+)1-bovine calbindin D9k.

The molecular basis for the co-operativity in binding of calcium ions by bovine calbindin D9k has been addressed by carrying out a comparative analysis of the solution conformation and dynamics of the apo, half saturated and fully saturated species using two-dimensional 1H nuclear magnetic resonance spectroscopy. Since the half saturated calcium form of the protein is not significantly populated under equilibrium conditions due to the co-operativity in binding of calcium ions, the half saturated cadmium form of the protein has been substituted for the calcium form. To verify that cadmium forms of calbindin D9k represent viable models for the calcium-bound species, the fully saturated cadmium form has been prepared and compared to the calcium-saturated protein. Virtually complete 1H resonance assignments have been obtained for both the (Cd2+)1 and the (Cd2+)2 states. Secondary structure elements and the global folding pattern were determined from nuclear Overhauser effects, backbone spin-spin coupling constants and slowly exchanging amide protons. Comparisons of the half saturated protein with the apo and calcium-saturated forms of calbindin D9k show that all three structures are highly similar. However, a change in the structural and dynamic properties of the protein does occur upon binding of the first ion; the half saturated form is found to be more similar to the calcium-saturated form than to the apo form. These results have important implications concerning the molecular basis for the co-operativity, and suggest that entropic effects associated with the protein dynamics play an important role.

Backbone dynamics and energetics of a calmodulin domain mutant exchanging between closed and open conformations.

Previous studies have suggested that the Ca2+-saturated E140Q mutant of the C-terminal domain of calmodulin exhibits equilibrium exchange between "open" and "closed" conformations similar to those of the Ca2+-free and Ca2+-saturated states of wild-type calmodulin. The backbone dynamics of this mutant were studied using15N spin relaxation experiments at three different temperatures. Measurements at each temperature of the15N rate constants for longitudinal and transverse auto-relaxation, longitudinal and transverse cross-correlation relaxation, and the1H-15N cross-relaxation afforded unequivocal identification of conformational exchange processes on microsecond to millisecond time-scales, and characterization of fast fluctuations on picosecond to nanosecond time-scales using model-free approaches. The results show that essentially all residues of the protein are involved in conformational exchange. Generalized order parameters of the fast internal motions indicate that the conformational substates are well folded, and exclude the possibility that the exchange involves a significant population of unfolded or disordered species. The temperature dependence of the order parameters offers qualitative estimates of the contribution to the heat capacity from fast fluctuations of the protein backbone, revealing significant variation between the well-ordered secondary structure elements and the more flexible regions. The temperature dependence of the conformational exchange contributions to the transverse auto-relaxation rate constants directly demonstrates that the microscopic exchange rate constants are greater than 2.7x10(3)s-1at 291 K. The conformational exchange contributions correlate with the chemical shift differences between the Ca2+-free and Ca2+-saturated states of the wild-type protein, thereby substantiating that the conformational substates are similar to the open and closed states of wild-type calmodulin. Taking the wild-type chemical shifts to represent the conformational substates of the mutant and populations estimated previously, the microscopic exchange rate constants could be estimated as 2x10(4)to 3x10(4)s-1at 291 K for a subset of residues. The temperature depen dence of the exchange allows the characterization of apparent energy barriers of the conformational transition, with results suggesting a complex process that does not correspond to a single global transition between substates.

Structural dynamics in the C-terminal domain of calmodulin at low calcium levels.

Calmodulin undergoes Ca2+-induced structural rearrangements that are intimately coupled to the regulation of numerous cellular processes. The C-terminal domain of calmodulin has previously been observed to exhibit conformational exchange in the absence of Ca2+. Here, we characterize further the conformational dynamics in the presence of low concentrations of Ca2+ using 15N spin relaxation experiments. The analysis included 1H-15N dipolar/15N chemical shift anisotropy interference cross-correlation relaxation rates to improve the description of the exchange processes, as well as the picosecond to nanosecond dynamics. Conformational transitions on microsecond to millisecond time scales were revealed by exchange contributions to the transverse auto-relaxation rates. In order to separate the effects of Ca2+ exchange from intramolecular conformational exchange processes in the apo state, transverse auto-relaxation rates were measured at different concentrations of free Ca2+. The results reveal a Ca2+-dependent contribution due mainly to exchange between the apo and (Ca2+)1 states with an apparent Ca2+ off-rate of approximately 5115 s(-1), as well as Ca2+-independent contributions due to conformational exchange within the apo state. 15N chemical shift differences estimated from the exchange data suggest that the first Ca2+ binds preferentially to loop IV. Thus, characterization of chemical exchange as a function of Ca2+ concentration has enabled the extraction of unique information on the rapidly exchanging and weakly populated (<10 %) (Ca2+)1 state that is otherwise inaccessible to direct study due to strongly cooperative Ca2+ binding. The conformational exchange within the apo state appears to involve transitions between a predominantly populated closed conformation and a smaller population of more open conformations. The picosecond to nanosecond dynamics of the apo state are typical of a well-folded protein, with reduced amplitudes of motions in the helical segments, but with significant flexibility in the Ca2+-binding loops. Comparisons with order parameters for skeletal troponin C and calbindin D9k reveal key structural and dynamical differences that correlate with the different Ca2+-binding properties of these proteins.

High-resolution structure of calcium-loaded calbindin D9k.

The three-dimensional solution structure of calcium-loaded calbindin D9k has been determined using experimental constraints obtained from nuclear magnetic resonance spectroscopy. A total of 1176 constraints (16 per residue overall, 32 per residue for the core residues) was used for the final refinement, including 1002 distance and 174 dihedral angle constraints. In addition, 23 hydrogen bond constraints were used for the generation of initial structures. Stereospecific assignments were made for 37 of 61 (61%) prochiral methylene protons and the methyl groups of all three valine residues and five out of 12 leucine residues. These constraints were used as input for a series of calculations of three-dimensional structures using a combination of distance geometry and restrained molecular dynamics. The 33 best structures selected for further analysis have no distance constraint violations greater than 0.3 A and good local geometries as reflected by low total energies (< or = -1014 kcal/mol in the AMBER 4.0 force field). The core of the protein consists of four well-defined helices with root-mean-square deviations from the average of 0.45 A for the N, C alpha and C' backbone atoms. These helices are packed in an antiparallel fashion to form two helix-loop-helix calcium-binding motifs, termed EF-hands. The two EF-hands are joined at one end by a ten-residue linker segment, and at the other by a short beta-type interaction between the two calcium-binding loops. Overall, the average solution structure of calbindin D9k is very similar to the crystal structure, with a pairwise root-mean-square deviation of 0.85 A for the N, C alpha and C' backbone atoms of the four helices. The differences that are observed between the solution and the crystal structures are attributed to specific crystal contacts, increased side-chain flexibility in solution, or artifacts arising from molecular dynamics refinement of the solution structures in vacuo.

Nuclear magnetic resonance studies of the internal dynamics in Apo, (Cd2+)1 and (Ca2+)2 calbindin D9k. The rates of amide proton exchange with solvent.

The backbone dynamics of the EF-hand Ca(2+)-binding protein, calbindin D9k, has been investigated in the apo, (Cd2+)1 and (Ca2+)2 states by measuring the rate constants for amide proton exchange with solvent. 15N-1H correlation spectroscopy was utilized to follow direct 1H-->2H exchange of the slowly exchanging amide protons and to follow indirect proton exchange via saturation transfer from water to the rapidly exchanging amide protons. Plots of experimental rate constants versus intrinsic rate constants have been analyzed to give qualitative insight into the opening modes of the protein that lead to exchange. These results have been interpreted within the context of a progressive unfolding model, wherein hydrophobic interactions and metal chelation serve to anchor portions of the protein, thereby damping fluctuations and retarding amide proton exchange. The addition of Ca2+ or Cd2+ was found to retard the exchange of many amide protons observed to be in hydrogen-bonding environments in the crystal structure of the (Ca2+)2 state, but not of those amide protons that were not involved in hydrogen bonds. The largest changes in rate constant occur for residues in the ion-binding loops, with substantial effects also found for the adjacent residues in helices I, II and III, but not helix IV. The results are consistent with a reorganization of the hydrogen-bonding networks in the metal ion-binding loops, accompanied by a change in the conformation of helix IV, as metal ions are chelated. Further analysis of the results obtained for the three states of metal occupancy provides insight into the nature of the changes in conformational fluctuations induced by ion binding.

15N NMR assignments and chemical shift analysis of uniformly labeled 15N calbindin D9k in the apo, (Cd2+)1 and (Ca2+)2 states.

15N has been uniformly incorporated into the EF-hand Ca(2+)-binding protein calbindin D9k so that heteronuclear experiments can be used to further characterize the structure and dynamics of the apo, (Cd2+)1 and (Ca2+)2 states of the protein. The 15N NMR resonances were assigned by 2D 15N-resolved 1H experiments, which also allowed the identification of a number of sequential and medium-range 1H-1H contacts that are obscured by chemical shift degeneracy in homonuclear experiments. The 15N chemical shifts are analyzed with respect to correlations with protein secondary structure. In addition, the changes in 15N chemical shift found for the apo----(Cd2+)1----(Ca2+)2 binding sequence confirm that the effects on the protein are mainly associated with chelation of the first ion.

May the driving force be with you--whatever it is.

Changes in the atomic coordinate fluctuations contribute to the entropy of biomolecular processes such as complex formation. Characterizations of such changes in proteins reveal that the response may be dramatically different between the backbone and the side chains, and further resolve enthalpy-entropy compensation at the molecular level.

Protein stability and electrostatic interactions between solvent exposed charged side chains.

To investigate the contribution to protein stability of electrostatic interactions between charged surface residues, we have studied the effect of substituting three negatively charged solvent exposed residues with their side-chain amide analogs in bovine calbindin D9k--a small (Mr 8,500) globular protein of the calmodulin superfamily. The free energy of urea-induced unfolding for the wild-type and seven mutant proteins has been measured. The mutant proteins have increased stability towards unfolding relative to the wild-type. The experimental results correlate reasonably well with theoretically calculated relative free energies of unfolding and show that electrostatic interactions between charges on the surface of a protein can have significant effects on protein stability.

Dynamics of ribonuclease H: temperature dependence of motions on multiple time scales.

The temperature dependence of the backbone motions in Escherichia coli ribonuclease HI was studied on multiple time scales by 15N nuclear magnetic spin relaxation. Laboratory frame relaxation data at 285, 300, and 310 K were analyzed using the model-free and reduced spectral density approaches. The temperature dependence of the order parameters was used to define a characteristic temperature for the motions of the backbone N-H bond vectors on picosecond to nanosecond time scales. The characteristic temperatures for secondary structure elements, loops, and the C-terminus are approximately 1000, approximately 300, and approximately 170 K, respectively. The observed variation in the characteristic temperature indicates that the energy landscape, and thus the configurational heat capacity, is markedly structure dependent in the folded protein. The effective correlation times for internal motions do not show significant temperature dependence. Conformational exchange was observed for a large number of residues forming a contiguous region of the protein that includes the coiled coil formed by helices alpha A and alpha D. Exchange broadening in the CPMG experiments decreased with increased temperature, directly demonstrating that the microscopic exchange rate is faster than the pulse repetition rate of 1.2 ms. The temperature dependence of the exchange contributions to the transverse relaxation rate constant shows approximately Arrhenius behavior over the studied temperature range with apparent activation enthalpies of approximately 20-50 kJ/mol. Numerical calculations suggest that these values underestimate the activation barriers by at most a factor of 2. The present results obtained at 300 K are compared to those reported previously [Mandel, A. M., Akke, M., & Palmer, A. G., III (1995) J. Mol. Biol. 246, 144-163] to establish the reproducibility of the experimental techniques.

From snapshot to movie: phi analysis of protein folding transition states taken one step further.

Kinetic anomalies in protein folding can result from changes of the kinetic ground states (D, I, and N), changes of the protein folding transition state, or both. The 102-residue protein U1A has a symmetrically curved chevron plot which seems to result mainly from changes of the transition state. At low concentrations of denaturant the transition state occurs early in the folding reaction, whereas at high denaturant concentration it moves close to the native structure. In this study we use this movement to follow continuously the formation and growth of U1A's folding nucleus by phi analysis. Although U1A's transition state structure is generally delocalized and displays a typical nucleation-condensation pattern, we can still resolve a sequence of folding events. However, these events are sufficiently coupled to start almost simultaneously throughout the transition state structure.

Three-dimensional solution structure of Ca(2+)-loaded porcine calbindin D9k determined by nuclear magnetic resonance spectroscopy.

The three-dimensional solution structure of native, intact porcine calbindin D9k has been determined by distance geometry and restrained molecular dynamics calculations using distance and dihedral angle constraints obtained from 1H NMR spectroscopy. The protein has a well-defined global fold consisting of four helices oriented in a pairwise antiparallel manner such that two pairs of helix-loop-helix motifs (EF-hands) are joined by a linker segment. The two EF-hands are further coupled through a short beta-type interaction between the two Ca(2+)-binding loops. Overall, the structure is very similar to that of the highly homologous native, minor A form of bovine calbindin D9k determined by X-ray crystallography [Szebenyi, D. M. E., & Moffat, K. (1986) J. Biol. Chem. 261, 8761-8776]. A model structure built from the bovine calbindin D9k crystal structure shows several deviations larger than 2 A from the experimental distance constraints for the porcine protein. These structural differences are efficiently removed by subjecting the model structure to the experimental distance and dihedral angle constraints in a restrained molecular dynamics protocol, thereby generating a model that is very similar to the refined distance geometry derived structures. The N-terminal residues of the intact protein that are absent in the minor A form appear to be highly flexible and do not influence the structure of other regions of the protein. This result is important because it validates the conclusions drawn from the wide range of studies that have been carried out on minor A forms rather than the intact calbindin D9k.

Effects of ion binding on the backbone dynamics of calbindin D9k determined by 15N NMR relaxation.

The backbone dynamics of apo- and (Cd2+)1-calbindin D9k have been characterized by 15N nuclear magnetic resonance spectroscopy. Spin-lattice and spin-spin relaxation rate constants and steady-state [1H]-15N nuclear Overhauser effects were measured at a magnetic field strength of 11.74 T by two-dimensional, proton-detected heteronuclear NMR experiments using 15N-enriched samples. The relaxation parameters were analyzed using a model-free formalism that characterizes the dynamics of the N-H bond vectors in terms of generalized order parameters and effective correlation times. The data for the apo and (Cd2+)1 states were compared to those for the (Ca2+)2 state [Kördel, J., Skelton, N. J., Akke, M., Palmer, A. G., & Chazin, W. J. (1992) Biochemistry 31, 4856-4866] to ascertain the effects on ion ligation on the backbone dynamics of calbindin D9k. The two binding loops respond differently to ligation by metal ions: high-frequency (10(9)-10(12) s-1) fluctuations of the N-terminal ion-binding loop are not affected by ion binding, whereas residues G57, D58, G59, and E60 in the C-terminal ion-binding loop have significantly lower order parameters in the apo state than in the metal-bound states. The dynamical responses of the four helices to binding of ions are much smaller than that for the C-terminal binding loop, with the strongest effect on helix III, which is located between the linker loop and binding site II. Significant fluctuations on slower time scales also were detected in the unoccupied N-terminal ion-binding loop of the apo and (Cd2+)1 states; the apparent rates were greater for the (Cd2+)1 state. These results on the dynamical response to ion binding in calbindin D9k provide insights into the molecular details of the binding process and qualitative evidence for entropic contributions to the cooperative phenomenon of calcium binding for the pathway in which the ion binds first in the C-terminal site.

Base dynamics in a UUCG tetraloop RNA hairpin characterized by 15N spin relaxation: correlations with structure and stability.

Intramolecular dynamics of guanine and uracil bases in a 14-nt RNA hairpin including the extraordinarily stable UUCG tetraloop were studied by 15N spin relaxation experiments that are sensitive to structural fluctuations occurring on a time scale of picoseconds to nanoseconds. The relaxation data were interpreted in the framework of the anisotropic model-free formalism, using assumed values for the chemical shift anisotropies of the 15N spins. The rotational diffusion tensor was determined to be symmetric with an axial ratio of 1.34 +/- 0.12, in agreement with estimates based on the ratio of the principal moments of the inertia tensor. The model-free results indicate that the bases of the G x U pair in the tetraloop are at least as rigid as the interior base pairs in the stem, whereas the 5'-terminal guanine is more flexible. The observed range of order parameters corresponds to base fluctuations of 19-22 degrees about the chi torsion angle. The results reveal dynamical consequences of the unusual structural features in the UUCG tetraloop and offer insights into the configurational entropy of hairpin formation.

Signal transduction versus buffering activity in Ca(2+)-binding proteins.

The three-dimensional structure of calbindin D9k in the absence of Ca2+ has been determined using NMR spectroscopy in solution, allowing the first direct analysis of the consequences of Ca2+ binding for a member of the calmodulin superfamily of proteins. The overall response in calbindin D9k is much attenuated relative to the current model for calmodulin and troponin C. These results demonstrate a novel mechanism for modulating the conformational response to Ca(2+)-binding in calmodulin superfamily proteins and provide insights into how their Ca(2+)-binding domains can be fine-tuned to remain essentially intact or respond strongly to ion binding, in relation to their functional requirements.