Conformational changes in C1q upon binding to IgG oligomers

The interaction between C1q and immune complexes is inhibited by 1-anilino-8-naphthalenesulfonate (ANS) in the concentration range of 2-4 mM. ANS binds to Clq with a 20-fold higher affinity than to IgG [(1986) Mol. Immunol. 23, 39-44] and therefore it is possible to label only C1q with ANS in the presence of IgG. Under such conditions no inhibition is observed. Addition of monomer IgG to a solution of C1q-bound ANS did not significantly alter the fluorescence of the ANS. However when oligomeric IgG was added there was a 2-fold increase in fluorescence over the same IgG concentration range. When C1q was pretreated with diethylpyrocarbonate there was little change in the fluorescence when IgG oligomers were added to C1q:ANS solutions. These results suggest that C1q undergoes conformational changes upon binding to IgG oligomers.     

Conformational changes and complement activation induced upon antigen binding to antibodies

The interaction between antibodies specific for the loop region of lysozyme and this monovalent antigenic determinant was compared to their interaction with either the dimeric derivative of the loop (bis-loop) or the polyvalent antigen loop-A--L. This comparison was based on complement fixation capacity, and on spectroscopic changes in the circular polarized fluorescence (CPL) of the antibodies. The binding of the loop to its antibodies causes spectroscopic changes, assigned to conformational changes in the Fc region, which are not accompanied by complement activation. However, the binding of the bisloop led to distinctly different CPL changes and concomitantly to complement fixation comparable to that induced upon binding of the loop-A--L. Complement fixation and the CPL changes can be induced in the intact antibodies only; reduction of the interchain disulfide bridges, or cleavage by papain or pepsin digestion abolishes both activities.     

Helix A stabilization precedes amino-terminal lobe activation upon calcium binding to calmodulin

The structural coupling between opposing domains of CaM was investigated using the conformationally sensitive biarsenical probe 4,5-bis(1,3,2-dithioarsolan-2-yl)resorufin (ReAsH), which upon binding to an engineered tetracysteine motif near the end of helix A (Thr-5 to Phe-19) becomes highly fluorescent. Changes in conformation and dynamics are reflective of the native CaM structure, as there is no change in the (1)H- (15)N HSQC NMR spectrum in comparison to wild-type CaM. We find evidence of a conformational intermediate associated with CaM activation, where calcium occupancy of sites in the amino-terminal and carboxyl-terminal lobes of CaM differentially affect the fluorescence intensity of bound ReAsH. Insight into the structure of the conformational intermediate is possible from a consideration of calcium-dependent changes in rates of ReAsH binding and helix A mobility, which respectively distinguish secondary structural changes associated with helix A stabilization from the tertiary structural reorganization of the amino-terminal lobe of CaM necessary for high-affinity binding to target proteins. Helix A stabilization is associated with calcium occupancy of sites in the carboxyl-terminal lobe ( K d = 0.36 +/- 0.04 microM), which results in a reduction in the rate of ReAsH binding from 4900 M (-1) s (-1) to 370 M (-1) s (-1). In comparison, tertiary structural changes involving helix A and other structural elements in the amino-terminal lobe require calcium occupancy of amino-terminal sites (K d = 18 +/- 3 microM). Observed secondary and tertiary structural changes involving helix A in response to the sequential calcium occupancy of carboxyl- and amino-terminal lobe calcium binding sites suggest an important involvement of helix A in mediating the structural coupling between the opposing domains of CaM. These results are discussed in terms of a model in which carboxyl-terminal lobe calcium activation induces secondary structural changes within the interdomain linker that release helix A, thereby facilitating the formation of calcium binding sites in the amino-terminal lobe and linked tertiary structural rearrangements to form a high-affinity binding cleft that can associate with target proteins.     

Phosphorylation-dependent binding of a synthetic MARCKS peptide to calmodulin

A 25-amino acid peptide, containing the four protein kinase C (PKC) phosphorylation sites and the calmodulin (CaM) binding domain of the myristoylated alanine-rich C kinase substrate (MARCKS) protein, has been synthesized and used to determine the effects of phosphorylation on its binding and regulation of CaM. PKC phosphorylation of this peptide (3.0 mol of Pi/mol of peptide) produced a 200-fold decrease in its affinity for CaM. PKC phosphorylation of the peptide resulted in its dissociation from CaM over a time course that paralleled the phosphorylation of 1 mol of serine/mol of peptide. The peptide inhibited CaM's binding to myosin light chain kinase and CaM's stimulation of phosphodiesterase and calcineurin. PKC phosphorylation of the peptide resulted in a rapid release of bound CaM, allowing its subsequent binding to myosin light chain kinase (t1/2 = 1.6 min), stimulation of phosphodiesterase (t1/2 = 1.2 min) and calcineurin (t1/2 = 1.7 min). Partially purified MARCKS protein produced a similar inhibition of CaM-phosphodiesterase which was reversed by PKC phosphorylation. PKC phosphorylation of the peptide occurred primarily at serine 8 and serine 12, and phosphorylation of serine 12 regulated peptide affinity for CaM. Thus, PKC phosphorylation of the peptide and the MARCKS protein results in the rapid release of CaM and the subsequent activation of CaM-dependent enzymes. This process might allow for interplay between PKC and CaM-dependent signal transduction pathways.     

Conformational changes in dopamine transporter intracellular regions upon cocaine binding and dopamine translocation

The dopamine transporter (DAT), a member of the neurotransmitter:sodium symporter family, mediates the reuptake of dopamine at the synaptic cleft. DAT is the primary target for psychostimulants such as cocaine and amphetamine. We previously demonstrated that cocaine binding and dopamine transport alter the accessibility of Cys342 in the third intracellular loop (IL3). To study the conformational changes associated with the functional mechanism of the transporter, we made cysteine substitution mutants, one at a time, from Phe332 to Ser351 in IL3 of the background DAT construct, X7C, in which 7 endogenous cysteines were mutated. The accessibility of the 20 engineered cysteines to polar charged sulfhydryl reagents was studied in the absence and presence of cocaine or dopamine. Of the 11 positions that reacted with methanethiosulfonate ethyl ammonium, as evidenced by inhibition of ligand binding, 5 were protected against this inhibition by cocaine and dopamine (S333C, S334C, N336C, M342C and T349C), indicating that reagent accessibility is affected by conformational changes associated with inhibitor and substrate binding. In some of the cysteine mutants, transport activity is disrupted, but can be rescued by the presence of zinc, most likely because the distribution between inward- and outward-facing conformations is restored by zinc binding. The experimental data were interpreted in the context of molecular models of DAT in both the inward- and outward-facing conformations. Differences in the solvent accessible surface area for individual IL3 residues calculated for these states correlate well with the experimental accessibility data, and suggest that protection by ligand binding results from the stabilization of the outward-facing configuration. Changes in the residue interaction networks observed from the molecular dynamics simulations also revealed the critical roles of several positions during the conformational transitions. We conclude that the IL3 region of DAT undergoes significant conformational changes in transitions necessary for both cocaine binding and substrate transport.     

A synthetic 33-residue analogue of bovine brain calmodulin calcium binding site III: synthesis, purification, and calcium binding

The sequential solid-phase synthesis of a peptide analogue of bovine brain calmodulin calcium binding site III covering residues 81-113 of the natural sequence is described. Methionine-109 is replaced by a leucine residue to avoid complications in the synthesis and purification. In an attempt to relate the structure of the calcium binding sites in the naturally occurring calcium binding protein to the calcium affinity of these sites, the synthetic analogue is examined for calcium binding by circular dichroism spectroscopy. The calcium binding characteristics are compared to those of a synthetic analogue of the homologous calcium binding site III in rabbit skeletal troponin C. The Kd of the calmodulin site III fragment for Ca2+ is determined as 878 microM whereas the Kd of the troponin C fragment is 30 times smaller at 28 microM. Structural changes induced in the peptides by Ca2+ and trifluoroethanol are similar. This study supports our contention that the single synthetic calcium binding site is a reasonable model for the study of the structure-activity relationships of the calcium binding sites in calcium-regulated proteins such as calmodulin and troponin C.     

Effect of phosphorylation of calmodulin on calcium binding affinity as estimated by terbium fluorescence

The effect of phosphorylation of calmodulin by casein kinase 2 on the calcium binding of the former was studied by measurement of terbium fluorescence. The binding of Tb3+ to calmodulin was followed by an increase in Tb3+ fluorescence at 545 nm. The terbium fluorescence of phosphorylated calmodulin increased at a lower concentration of Tb3+ than that of non-phosphorylated calmodulin, indicating that Tb3+ binding affinity of calmodulin was increased by phosphorylation. Our results suggest that the interaction between calcium and binding domain becomes stronger by phosphorylation.     

Conformational changes in tubulin upon binding cryptophycin-52 reveal its mechanism of action

Cryptophycin-52 (Cp-52) is potentially the most potent anticancer drug known, with IC₅₀ values in the low picomolar range, but its binding site on tubulin and mechanism of action are unknown. Here, we have determined the binding site of Cp-52, and its parent compound, cryptophycin-1, on HeLa tubulin, to a resolution of 3.3 Å and 3.4 Å, respectively, by cryo-EM and characterized this binding further by molecular dynamics simulations. The binding site was determined to be located at the tubulin interdimer interface and partially overlap that of maytansine, another cytotoxic tubulin inhibitor. Binding induces curvature both within and between tubulin dimers that is incompatible with the microtubule lattice. Conformational changes occur in both α-tubulin and β-tubulin, particularly in helices H8 and H10, with distinct differences between α and β monomers and between Cp-52-bound and cryptophycin-1-bound tubulin. From these results, we have determined: (i) the mechanism of action of inhibition of both microtubule polymerization and depolymerization, (ii) how the affinity of Cp-52 for tubulin may be enhanced, and (iii) where linkers for targeted delivery can be optimally attached to this molecule.     

Conformational changes of colicin Ia channel-forming domain upon membrane binding: a solid-state NMR study

Channel-forming colicins are bactericidal proteins that spontaneously insert into hydrophobic lipid bilayers. We have used magic-angle spinning solid-state nuclear magnetic resonance spectroscopy to examine the conformational differences between the water-soluble and the membrane-bound states of colicin Ia channel domain, and to study the effect of bound colicin on lipid bilayer structure and dynamics. We detected (13)C and (15)N isotropic chemical shift differences between the two forms of the protein, which indicate structural changes of the protein due to membrane binding. The Val C(alpha) signal, unambiguously assigned by double-quantum experiments, gave a 0.6 ppm downfield shift in the isotropic position and a 4 ppm reduction in the anisotropic chemical shift span after membrane binding. These suggest that the alpha-helices in the membrane-bound colicin adopt more ideal helical torsion angles as they spread onto the membrane. Colicin binding significantly reduced the lipid chain order, as manifested by (2)H quadrupolar couplings. These results are consistent with the model that colicin Ia channel domain forms an extended helical array at the membrane-water interface upon membrane binding.     

Conformational changes in arginine kinase upon ligand binding seen by small-angle X-ray scattering

Small-angle X-ray scattering is used to study the effects of substrate binding to lobster arginine kinase in solution. We measure the radius of gyration of the enzyme in the absence and in the presence of ligands. We find that the radius of gyration decreases by 1.20 ± 0.25 Å upon binding ADP-Mg and L-arginine to form the ternary complex. The same decrease is also observed upon binding ADP-Mg alone or ATP-Mg. These results indicate a large conformational change consistent with the hinge motion of domains observed in other phosphokinases.     

Conformational changes of colicin Ia channel-forming domain upon membrane binding: a solid-state NMR study

Channel-forming colicins are bactericidal proteins that spontaneously insert into hydrophobic lipid bilayers. We have used magic-angle spinning solid-state nuclear magnetic resonance spectroscopy to examine the conformational differences between the water-soluble and the membrane-bound states of colicin Ia channel domain, and to study the effect of bound colicin on lipid bilayer structure and dynamics. We detected ¹³C and ¹⁵N isotropic chemical shift differences between the two forms of the protein, which indicate structural changes of the protein due to membrane binding. The Val Cα signal, unambiguously assigned by double-quantum experiments, gave a 0.6 ppm downfield shift in the isotropic position and a 4 ppm reduction in the anisotropic chemical shift span after membrane binding. These suggest that the α-helices in the membrane-bound colicin adopt more ideal helical torsion angles as they spread onto the membrane. Colicin binding significantly reduced the lipid chain order, as manifested by ²H quadrupolar couplings. These results are consistent with the model that colicin Ia channel domain forms an extended helical array at the membrane-water interface upon membrane binding.     

Conformational changes in the bacterial SRP receptor FtsY upon binding of guanine nucleotides and SRP

In cotranslational preprotein targeting in Escherichia coli, the signal recognition particle (SRP) binds to the signal peptide emerging from the ribosome and, subsequently, interacts with the signal recognition particle receptor, FtsY, at the plasma membrane. Both FtsY and the protein moiety of the signal recognition particle, Ffh, are GTPases, and GTP is required for the formation of the SRP-FtsY complex. We have studied the binding of GTP/GDP to FtsY as well as the SRP-FtsY complex formation by monitoring the fluorescence of tryptophan 343 in the I box of mutant FtsY. Thermodynamic and kinetic parameters of the FtsY complexes with GDP, GTP, and signal recognition particle are reported. Upon SRP-FtsY complex formation in the presence of GTP, the fluorescence of tryptophan 343 increased by 50 % and was blue-shifted by 10 nm. We conclude that GTP-dependent SRP-FtsY complex formation leads to an extensive conformational change in the I box insertion in the effector region of FtsY.     

Effects of trifluoperazine on calcium binding by calmodulin. Heat capacity and entropy changes

The possible structural changes of the calmodulin-trifluoperazine (TFP) complex caused by Ca2+ binding have been analyzed by microcalorimetric titrations. Titrations of calmodulin with Ca2+ in the presence of 8-fold molar excess TFP have been made both in the absence and presence of Mg2+, at pH 7.0, and at 5, 15, and 25 degrees C. At high concentrations of TFP calmodulin forms a complex with TFP even in the absence of Ca2+. The reaction of the calmodulin-TFP complex with Ca2+ is exothermic, both in the presence and absence of Mg2+. In the presence of Mg2+ the reaction is driven almost entirely by a favorable enthalpy change. The magnitudes of the hydrophobic and internal vibrational contributions to the heat capacity and entropy changes of this complex on Ca2+ binding have been estimated by the empirical method of Sturtevant (Sturtevant, J. M. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 2236-2240). In the presence of Mg2+, the vibrational as well as hydrophobic entropy is slightly increased in a parallel manner by Ca2+ binding to each of the binding sites. In contrast, when Mg2+ is absent, the hydrophobic entropy gradually increases on Ca2+ binding, but the vibrational entropy decreases. These changes of entropy indicate the assembling of non-polar groups on the surface of the complex and suggest that the overall structure is loosened in the presence of Mg2+, but tightened in the absence of Mg2+.     

Brain spectrin binding to the NMDA receptor is regulated by phosphorylation, calcium and calmodulin.

The N-methyl-D-aspartate receptor (NMDA-R) and brain spectrin, a protein that links membrane proteins to the actin cytoskeleton, are major components of post-synaptic densities (PSDs). Since the activity of the NMDA-R channel is dependent on the integrity of actin and leads to calpain-mediated spectrin breakdown, we have investigated whether the actin-binding spectrin may interact directly with NMDA-Rs. Spectrin is reported here to interact selectively in vitro with the C-terminal cytoplasmic domains of the NR1a, NR2A and NR2B subunits of the NMDA-R but not with that of the AMPA receptor GluR1. Spectrin binds at NR2B sites distinct from those of alpha-actinin-2 and members of the PSD95/SAP90 family. The spectrin-NR2B interactions are antagonized by Ca2+ and fyn-mediated NR2B phosphorylation, but not by Ca2+/calmodulin (CaM) or by Ca2+/CaM-dependent protein kinase II-mediated NR2B phosphorylation. The spectrin-NR1 interactions are unaffected by Ca2+ but inhibited by CaM and by protein kinase A- and C-mediated phosphorylations of NR1. Finally, in rat synaptosomes, both spectrin and NR2B are loosened from membranes upon addition of physiological concentrations of calcium ions. The highly regulated linkage of the NMDA-R to spectrin may underlie the morphological changes that occur in neuronal dendrites concurrently with synaptic activity and plasticity.     

Peptide binding by a fragment of calmodulin composed of EF-hands 2 and 3

Calmodulin (CaM) is composed of two EF-hand domains tethered by a flexible linker. Upon Ca2+-binding, a fragment of CaM encompassing EF-hands 2 and 3 (CaM2/3; residues 46-113) folds into a structure remarkably similar to the N- and C-domains of CaM. In this study, we demonstrate that Ca2+-ligated CaM2/3 can also bind to a peptide representing the CaM-recognition sequence of skeletal muscle myosin light chain kinase (M13) with an equimolar stoichiometry and a dissociation constant of 0.40 +/- 0.05 microM. On the basis of an analytical ultracentrifugation measurement, the resulting complex exists as an equilibrium mixture of 2:2 heterotetrameric and 1:1 heterodimeric species. Chemical shift perturbation mapping indicates that, similar to CaM, the peptide associates with a hydrophobic groove crossing both EF-hands in CaM2/3. However, upon binding the M13 peptide, many residues in CaM2/3 yielded two equal intensity NMR signals with the same 15N relaxation properties. Thus, the 2:2 CaM2/3-M13 tetramer, which predominates under the conditions used for these studies, is asymmetric with each component adopting spectroscopically distinguishable conformations within the complex. CaM2/3 also weakly stimulates the phosphatase activity of calcineurin and inhibits stimulation by native CaM. These studies highlight the remarkable plasticity of EF-hand association and expand the diverse repertoire of mechanisms possible for CaM-target protein interactions.     

Multiple calcium binding sites make calmodulin multifunctional

Protein-protein or protein-ion interactions with multisite proteins are essential to the regulation of intracellular and extracellular events. There is, however, limited understanding of how ligand-multisite protein interactions selectively regulate the activities of multiple protein targets. In this paper, we focus on the important calcium (Ca(2+)) binding protein calmodulin (CaM), which has four Ca(2+) ion binding sites and regulates the activity of over 30 other proteins. Recent progress in structural studies has led to significant improvements in the understanding of Ca(2+)-CaM-dependent regulation mechanisms. However, no quantitative model is currently available that can fully explain how the structural diversity of protein interaction surfaces leads to selective activation of protein targets. In this paper, we analyze the multisite protein-ligand binding mechanism using mathematical modelling and experimental data for Ca(2+)-CaM-dependent protein targets. Our study suggests a potential mechanism for selective and differential activation of Ca(2+)-CaM targets by the same CaM molecules, which are involved in a variety of intracellular functions. The close agreement between model predictions and experimental dose-response curves for CaM targets available in the literature suggests that such activation is due to the selective activity of CaM conformations in complexes with variable numbers of Ca(2+) ions. Although the paper focuses on the Ca(2+)-CaM pair as a particularly data rich example, the proposed model predictions are quite general and can easily be extended to other multisite proteins. The results of the study may therefore be proposed as a general explanation for multifunctional target regulation by multisite proteins.     

Conformational changes in the PBX homeodomain and C-terminal extension upon binding DNA and HOX-derived YPWM peptides

PBX is a member of the three amino acid loop extension (TALE) class of homeodomains. PBX binds DNA cooperatively with HOX homeodomain proteins that contain a conserved YPWM motif. The amino acids immediately C-terminal to the PBX homeodomain increase the affinity of the homeodomain for its DNA site and HOX proteins. We have determined the structure of the free PBX homeodomain using NMR spectroscopy. Both the PBX homeodomain and the extended PBX homeodomain make identical contacts with a 5'-TGAT-3' DNA site and a YPWM peptide. A fourth alpha-helix, which forms upon binding to DNA, stabilizes the extended PBX structure. Variations in DNA sequence selectivity of heterodimeric PBX-HOX complexes depend on the HOX partner; however, a comparison of five different HOX-derived YPWM peptides showed that each bound to PBX in the same way, differing only in the strength of the association.     

Proton nuclear magnetic resonance studies on the kinetics of tryptic fragments of calmodulin upon calcium binding

The kinetics of the Ca2+-dependent conformational change of the tryptic fragments F12 (residues 1-75) and F34 (residues 78-148) of calmodulin were studied by 1H-NMR. Resonances of two phenylalanines, 16 (or 19) and 65 (or 68), N epsilon, N epsilon, N epsilon-trimethyllysine-115 and tyrosine-138 were examined by the saturation-transfer technique or computer-aided line-shape simulation to obtain the rate of the conformational exchange between the Ca2+-free form and the Ca2+-bound form. The rates for F12 and F34 in the presence of 0.2 M KCl at 22 degrees C were 300-500 s-1 and 3-10 s-1, respectively. Activation parameters are as follows: Delta H not equal to = 11(+/- 2) kcal X M-1 and delta S not equal to = -9(+/- 5) cal X K-1 X M-1 for F12, and delta H not equal to = 16(+/- 2) kcal X M-1 and delta S not equal to = -2(+/- 5) cal X K-1 X M-1 for F34. These kinetic data for the conformational exchange are in agreement with those of Ca2+ dissociation from the binding sites obtained by 43Ca-NMR and stopped-flow fluorescence studies.