Binding and exchange of nucleotides on the chloroplast coupling factor CF1. The role of magnesium

On the soluble part of the coupling factor (CF1), extracted from spinach chloroplasts, three nucleotide-binding sites are identified. Three ADP are bound per CF1 when the enzyme is incubated with ADP either with or without Mg2+. Two ADP and one ATP are bound per CF1 when the enzyme is incubated with a limiting concentration of ATP, in the presence of Mg2+. At high ATP concentration, in the presence of Mg2+, one free ATP exchanges with one bound ADP and two ATP and one ADP remain bound per CF1. When Mg2+ is omitted from the incubation medium of ATP and CF1, only two ADP and around 0.5 ATP are bound per CF1. The three nucleotide binding sites of CF1 fall into two different and independent categories according to the ability of the bound nucleotides to be exchanged with free nucleotides. On one site the bound ADP is difficult to exchange. On the other two sites, the bound nucleotides. ADP or ATP, are readily exchangable. We propose that the two exchangeable sites form the catalytic part of the enzyme where ATP is hydrolyzed. When ATP concentration is high enough, in the presence of Mg2+, one ATP displaces one bound ADP and allows the ATP hydrolysis to proceed. We propose too that the site where ADP is difficult to exchange may represent the 'tight' ADP-binding site, different from the catalytic ones, which becomes exchangeable on the CF1 in vivo when the thylakoid membranes are energized by light, as stressed by Bickel-Sandk?tter and Strotman [(1976) FEBS Lett. 65, 102-106].     

113Cd nuclear magnetic resonance of Cd(II) alkaline phosphatases

113Cd NMR spectra of 113Cd(II)-substituted Escherichia coli alkaline phosphatase have been recorded over a range of pH values, levels of metal site occupancy, and states of phosphorylation. Under all conditions resonances attributable to cadmium specifically bound at one or more of the three pairs of metal-binding sites (A, B, and C sites) are detected. By following changes in both the 113Cd and 31P NMR spectra of 113Cd(II)2 alkaline phosphatase during and after phosphorylation, it has been possible to assign the cadmium resonance that occurs between 140 and 170 ppm to Cd(II) bound to the A or catalytic site of the enzyme and the resonance occurring between 51 and 76 ppm to Cd(II) bound to B site, which from x-ray data is located 3.9 A from the A site. The kinetics of phosphorylation show that cadmium migration from the A site of one subunit to the B site of the second subunit follows and is a consequence of phosphate binding, thus precluding the migration as a sufficient explanation for half-of-the-sites reactivity. Rather, there is evidence for subunit-subunit interaction rendering the phosphate binding sites inequivalent. Although one metal ion, at A site, is sufficient for phosphate binding and phosphorylation, the presence of a second metal ion at B site greatly enhances the rate of phosphorylation. In the absence of phosphate, occupation of the lower affinity B and C sites produces exchange broadening of the cadmium resonances. Phosphorylation abolishes this exchange modulation. Magnesium at high concentration broadens the resonances to the point of undetectability. The chemical shift of 113Cd(II) in both A and B sites (but not C site) is different depending on the state of the bound phosphate (whether covalently or noncovalently bound) and gives separate resonances for each form. Care must be taken in attributing the initial distribution of cadmium or phosphate in the reconstituted enzyme to that of the equilibrium species in samples reconstituted from apoenzyme. Both 113Cd NMR and 31P NMR show that some conformational changes consequent to metal ion or phosphate binding require several days before the final equilibrium species is formed.     

Nucleotide and AP5A complexes of porcine adenylate kinase: A 1H and 19F NMR study

Proton and fluorine nuclear magnetic resonance spectroscopies (NMR) were used as methods to investigate binary complexes between porcine adenylate kinase (AK1) and its substrates. We also studied the interaction of fluorinated substrate analogues and the supposed bisubstrate analogue P1,P5-bis(5'-adenosyl) pentaphosphate (AP5A) with AK1 in the presence of Mg2+. The chemical shifts of the C8-H, C2-H, and ribose C1'-H resonances of both adenosine units in stoichiometric complexes of AK1 with AP5A in the presence of Mg2+ could be determined. The C2-H resonance of one of the adenine bases experiences a downfield shift of about 0.8 ppm on binding to the enzyme. The chemical shift of the His36 imidazole C2-H was changed in the downfield direction on ATP-Mg2+ and, to a lesser extent, AMP binding. 19F NMR chemical shifts of 9-(3-fluoro-3-deoxy-beta-D-xylofuranosyl)adenine triphosphate (3'-F-X-ATP)-Mg2+ and 9-(3-fluoro-3-deoxy-beta-D-xylofuranosyl)adenine monophosphate (3'-F-X-AMP) bound to porcine adenylate kinase could be determined. The different chemical shifts of the bound nucleotides suggest that their mode of binding is different. Free and bound 3'-F-X-AMP are in fast exchange with respect to their 19F chemical shifts, whereas free and bound 3'-F-X-ATP are in slow exchange on the NMR time scale in the absence as well as in the presence of Mg2+. This information could be used to determine the apparent dissociation constants of the nucleotides and the 3'-F-X analogues in the binary complexes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cation binding in Na,K-ATPase, investigated by 205Tl solid-state NMR spectroscopy

Cation binding to Na,K-ATPase is characterized in native membranes at room temperature by solid-state NMR spectroscopy using the K(+) congener (205)Tl. It has been demonstrated that the signals from occluded Tl(+) and nonspecifically bound Tl(+) can be detected and distinguished by NMR. Effects of dipole-dipole coupling between (1)H and (205)Tl in the occlusion sites show that the ions are rigidly bound, rather than just occluded. Furthermore, a low chemical shift suggests occlusion site geometries with a relatively small contribution from carboxylate and hydroxyl groups. Nonspecific binding of Tl(+) is characterized by rapid chemical exchange, in agreement with the observed low binding affinity.     

Cre induces an asymmetric DNA bend in its target loxP site

Cre initiates recombination by preferentially exchanging the bottom strands of the loxP site to form a Holliday intermediate, which is then resolved on the top strands. We previously found that the scissile AT and GC base pairs immediately 5' to the scissile phosphodiester bonds are critical in determining this order of strand exchange. We report here that the scissile base pairs also influence the Cre-induced DNA bends, the position of which correlates with the initial site of strand exchange. The binding of one Cre molecule to a loxP site induces a approximately 35 degrees asymmetric bend adjacent to the scissile GC base pair. The binding of two Cre molecules to a loxP site induces a approximately 55 degrees asymmetric bend near the center of the spacer region with a slight bias toward the scissile A. Lys-86, which contacts the scissile nucleotides, is important for establishing the bend near the scissile GC base pair when one Cre molecule is bound but has little role in positioning the bend when two Cre molecules are bound to a loxP site. We present a model relating the position of the Cre-induced bends to the order of strand exchange in the Cre-catalyzed recombination reaction.     

Structural evidence for a functionally relevant second camphor binding site in P450cam: model for substrate entry into a P450 active site

P450cam has long served as a prototype for the cytochrome P450 (CYP) gene family. But, little is known about how substrate enters its active site pocket, and how access is achieved in a way that minimizes exposure of the reactive heme. We hypothesize that P450cam may first bind substrate transiently near the mobile F-G helix that covers the active site pocket. Such a two-step binding process is kinetically required if P450cam rarely populates an open conformation-as suggested by previous literature and the inability to obtain a crystal structure of P450cam in an open conformation. Such a mechanism would minimize exposure of the heme by allowing P450cam to stay in a closed conformation as long as possible, since only brief flexing into an open conformation would be required to allow substrate entry. To test this model, we have attempted to dock a second camphor molecule into the crystal structure of camphor-bound P450cam. The docking identified only one potential entry site pocket, a well-defined cavity on the F-helix side of the F-G flap, 16 A from the heme iron. Location of this entry site pocket is consistent with our NMR T1 relaxation-based measurements of distances for a camphor that binds in fast exchange (active site camphor is known to bind in slow exchange). Presence of a second camphor binding site is also confirmed with [(1)H-(13)C] HSQC titrations of (13)CH3-threonine labeled P450cam. To confirm that camphor can bind outside of the active site pocket, (13)CH3-S-pyridine was bound to the heme iron to physically block the active site, and to serve as an NMR chemical shift probe. Titration of this P450cam-pyridine complex confirms that camphor can bind to a site outside the active site pocket, with an estimated Kd of 43 microM. The two-site binding model that is proposed based on these data is analogous to that recently proposed for CYP3A4, and is consistent with recent crystal structures of P450cam bound to tethered-substrates, which force a partially opened conformation.     

Mechanism of the binding of Z-L-tryptophan and Z-L-phenylalanine to thermolysin and stromelysin-1 in aqueous solutions

The chemical shift of the carboxylate carbon of Z-tryptophan is increased from 179.85 to 182.82 ppm and 182.87 ppm on binding to thermolysin and stromelysin-1 respectively. The chemical shift of Z-phenylalanine is also increased from 179.5 ppm to 182.9 ppm on binding to thermolysin. From pH studies we conclude that the pK(a) of the inhibitor carboxylate group is lowered by at least 1.5 pK(a) units when it binds to either enzyme. The signal at ~183 ppm is no longer observed when the active site zinc atom of thermolysin or stromelysin-1 is replaced by cobalt. We estimate that the distance of the carboxylate carbon of Z-[1-(13)C]-L-tryptophan is ≤3.71? from the active site cobalt atom of thermolysin. We conclude that the side chain of Z-[1-(13)C]-L-tryptophan is not bound in the S(2)' subsite of thermolysin. As the chemical shifts of the carboxylate carbons of the bound inhibitors are all ~183 ppm we conclude that they are all bound in a similar way most probably with the inhibitor carboxylate group directly coordinated to the active site zinc atom. Our spectrophotometric results confirm that the active site zinc atom is tetrahedrally coordinated when the inhibitors Z-tryptophan or Z-phenylalanine are bound to thermolysin.     

Changes in structure and dynamics of the Fv fragment of a catalytic antibody upon binding of inhibitor

Binding of the product inhibitor p-nitrophenol to the monoclonal esterolytic antibody NPN43C9 has been investigated by performing NMR spectroscopy of the heterodimeric variable-domain fragment (Fv) of the antibody in the presence and absence of inhibitor. Structural information from changes in chemical shift upon binding has been related to the changes in local dynamics in the active site of the catalytic antibody using NMR relaxation measurements. Significant changes in the chemical shifts of the backbone resonances upon binding extend beyond the immediate vicinity of the antigen binding site into the interface between the two associated polypeptides that form the Fv heterodimer, a possible indication that the binding of ligand causes a change in the relative orientations of the component light (V(L)) and heavy (V(H)) chain polypeptides. Significant differences in backbone dynamics were observed between the free Fv and the complex with p-nitrophenol. A number of resonances, including almost all of the third hypervariable loop of the light chain (L3), were greatly broadened in the free form of the protein. Other residues in the antigen-binding site showed less broadening of resonances, but still required exchange terms (R(ex)) in the model-free dynamics analysis, consistent with motion on a slow timescale in the active site region of the free Fv. Binding of p-nitrophenol caused these resonances to sharpen, but some R(ex) terms are still required in the analysis of the backbone dynamics. We conclude that the slow timescale motions in the antigen-binding site are very different in the bound and free forms of the Fv, presumably due to the damping of large-amplitude motions by the bound inhibitor.     

Regulation of Sin recombinase by accessory proteins

Sin recombinase from Staphylococcus aureus acts selectively on directly repeated resH sites, assembling an intertwined synapse in which exactly three supercoils are trapped between the points of strand exchange. Resolution requires the two Sin binding sites in resH (site I, where strand exchange occurs, and site II) and a non-specific DNA-bending protein (e.g. Hbsu). We show that a single amino acid substitution in Sin (I100T) is sufficient to relax the normal requirements for site II and Hbsu. Using this hyperactive protein, and the variant recombination site resH(AT), we investigate the roles of site II and Hbsu in synapsis and strand exchange. We conclude that Sin bound at site II, and Hbsu, act together to control site I alignment and the topology of the synapse, and to stimulate strand exchange.     

Modeling of proton spin relaxation in muscle tissue using nuclear magnetic resonance spin grouping and exchange analysis.

NMR spin relaxation experiments performed on healthy mouse muscle tissue at 40 MHz and 293 K are reported. The spin-lattice relaxation experiments were performed using different combinations of selective and nonselective radio frequency pulses. Relaxation experiments in the rotating frame at H1 = 10, 5 and 1 G are also reported. The experimental results were analyzed using the spin-grouping method, which yields the sizes of the resolved magnetization components as well as their T2's and T1's (or T1p's) for the nonexponential relaxation functions. These results were analyzed further for the exchange between different spin groups. It has been found that to explain all of these experimental data it was necessary to use a four-compartment model of the muscle tissue that consists of a lipid spin group, a "solid-like" spin group (mainly proteins), a "bulk water" spin group and a "bound water" spin group. The chemical exchange rate between "bulk" and "bound" water was found to be 29 +/- 9s-1 at room temperature. The exchange rate between the bound water and the solid moderator was estimated to be approximately 500 s-1.     

Function of pyridoxal 5'-phosphate in glycogen phosphorylase: 19F NMR and kinetic studies of phosphorylase reconstituted with 6-fluoropyridoxal and 6-fluoropyridoxal phosphate

19F NMR spectroscopic properties of glycogen phosphorylase reconstituted with 6-fluoropyridoxal (6-FPAL) and 6-fluoropyridoxal phosphate (6-FPLP) were investigated. Analysis of the contribution of chemical shift anisotropy to the line width of the 6-FPLP-enzyme signal shows that the coenzyme molecule is tightly bound to the protein. The chemical shift of the fluorine nucleus in the free 6-FPLP protein is pH independent from pH 6 to pH 9.1. When the 6-FPLP-enzyme forms complexes with AMP, AMP plus glucose-1-P, and AMP plus inorganic phosphate, signals at -11.0, -13.1, and -10.4 ppm are observed, respectively. These different chemical shifts indicate that the protein in each complex has a distinct conformation. The exchange rate between the 6-FPLP-protein-AMP complex and the same complex with bound glucose-1-P is estimated to be 3300 +/- 700 s-1, and that between the 6-FPLP-protein-AMP complex and with bound inorganic phosphate is 500 +/- 100 s-1. The former exchange rate is 13 times faster than that of the same process for the 6-FPAL-enzyme. Analysis of the effects of temperature on the 19F line shape of the 6-FPLP enzyme in the presence of ligands shows that the exchange rates between different complexes drop significantly between 20 and 10 degrees C. Within this temperature range, Arrhenius plots of the enzymatic activities of the native and 6-FPLP-enzymes at varied temperatures also show a pronounced curvature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Decreased imino proton exchange and base-pair opening in the IHF-DNA complex measured by NMR.

Integration Host Factor, IHF, is an E. coli DNA binding protein that imposes a substantial bend on DNA. Previous footprinting studies and bending assays have characterized several recognition sequences in the bacterial and lambda phage genome as unique in the way they are bound by IHF. We have chosen one of the lambda phage sites, H1, for study because it presents a small yet sequence-specific substrate for NMR analysis of the complex. A 19 base-pair duplex, H19, corresponding to the recognition sequence at the H1 site was constructed by isotopically labeling one of the strands with 15N. (1H, 15N) heteronuclear NMR experiments aided in assigning the imino proton resonances of the DNA alone and in complex with IHF. The NMR results are consistent with a mode of binding observed in the recent crystal structure of IHF bound to another of its sites from the lambda phage genome. Additionally, the dramatic change that IHF imposes on the imino proton chemical shifts is indicative of a severe deviation from canonical B-DNA structure. In order to understand the dynamic properties of the DNA in the complex with IHF, the exchange rates of the imino protons with the solvent have been measured for H19 with and without IHF bound. A drastic reduction in exchange is observed for the imino protons in the IHF bound DNA. In the DNA-protein complex, groups of adjacent base-pair exchange at the same rate, and appear to close more slowly than the rate of imino proton exchange with bulk water, since their exchange rate is independent of catalyst concentration. We infer that segments of the double helix as large as 6 bp open in a cooperative process, and remain open much longer than is typical for opening fluctuations in naked duplex DNA. We discuss these results in terms of the specific protein-DNA contacts observed in the crystal structure.     

Complement subcomponent C1q secreted by cultured human monocytes has subunit structure identical with that of serum C1q.

Rat alpha-foetoprotein (alpha-FP) strongly binds the drugs warfarin and phenylbutazone, as does albumin; however, the binding sites for the two drugs seemed to be different. This possibility and the specificity of this/these drug-binding site(s) of rat alpha-FP were investigated by competitive protein-binding experiments with a variety of drugs, representing different pharmacological groups, and biomolecules that are strongly bound by the foetal protein and that are suspected to play a specific role during foetal development. The binding mechanisms were further investigated by using comparisons between computer-derived theoretical displacement curves and experimental points in order to distinguish different possible binding models. The results indicate: that warfarin and phenylbutazone are bound at two distinct sites on rat alpha-FP and that a negative modulatory effect is exerted between the two sites; that the degree of specificity of these two drug-binding sites is different, since the warfarin-binding site appears to be specific for the binding of coumarinic and anthranilic drugs whereas that for phenylbutazone is able to bind substances of very varied chemical structure and is more hydrophobic; that the phenylbutazone-binding site is the site that binds oestrogens that thyroid hormones and, probably, fatty acids and bilirubin are bound at (an)other site(s) but exert negative modulatory effects on phenylbutazone binding. The nature of the different binding areas of rat alpha-FP is compared with that of those already proposed for albumin. The potential risks of toxicity of such interactions between drugs and/or biomolecules on foetal development are also discussed.     

Analysis of the gel electrophoresis of looped protein-DNA complexes by computer simulation

The theory of mass transport coupled to reversible interactions under chemical kinetic control forms the basis of a numerical model that has been applied to systems such as lac repressor-lac operator DNA, in which a protein binds in two different modes to linear DNA carrying two specific binding sites. Three complexes may be formed: (1) a linear 1:1 complex with one protein molecule bound to one site on the DNA molecule; (2) a 1:1 complex in which a single protein molecule is bound to both sites simultaneously, thereby inducing a large DNA loop; and (3) a 2:1 linear complex in which two protein molecules are bound in tandem, each occupying a single site. The computational model affords a quantitative numerical simulation of the observed gel electrophoretic patterns produced by titration of the DNA with protein and provides new insights into the shape and nature of the patterns. In particular, the patterns may represent unimodal or bimodal reaction zones. Nevertheless, analysis of the peaks in the patterns obtained at low DNA and high protein concentration provides essential information as to the stoichiometry of the complexes and satisfactory estimates of association constants. The theory thus provides the experimenter with guidelines for quantitative evaluation of the results of gel retardation assays of the particular system under investigation, once protein-induced DNA (or RNA) loops have been established by independent physical or chemical methods. It is suggested that these insights might also find application to systems involving the binding of two or three different proteins to DNA with loop formation.     

Adenine nucleotide binding sites on beef heart F1-ATPase. Asymmetry and subunit location

Previously we have shown that beef heart mitochondrial F1 contains a total of six adenine nucleotide binding sites. Three "catalytic" sites exchange bound ligand rapidly during hydrolysis of MgATP, whereas three "noncatalytic" sites do not. The noncatalytic sites behave asymmetrically in that a single site releases bound ligand upon precipitation of F1 with ammonium sulfate. In the present study, we find this same site to be the only noncatalytic site that undergoes rapid exchange of bound ligand when F1 is incubated in the presence of EDTA at pH 8.0. Following 1000 catalytic turnovers/F1, the site retains the unique capacity for EDTA-induced exchange, indicating that the asymmetric determinants are permanent and that the three noncatalytic sites on soluble F1 do not pass through equivalent states during catalysis. Measurements of the rate of ligand binding at the unique noncatalytic site show that uncomplexed nucleotide binds preferentially. At pH 7.5, in the presence of Mg2+, the rate constant for ADP binding is 9 X 10(3) M-1 s-1 and for dissociation is 4 X 10(-4) s-1 to give a Kd = 50 nM. The rate of dissociation is 10 times faster in the presence of EDTA or during MgATP hydrolysis, and it increases rapidly at pH below 7. EDTA-induced exchange is inhibited by Mg2+, Mn2+, Co2+, and Zn2+ but not by Ca2+ and is unaffected by dicyclohexylcarbodiimide modification. The unique noncatalytic site binds 2-azido-ADP. Photolysis results in the labeling of the beta subunit. Photolabeling of a single high-affinity catalytic site under conditions for uni-site catalysis also results in the labeling of beta, but a different pattern of labeled peptides is obtained in proteolytic digests. The results demonstrate the presence of two different nucleotide binding domains on the beta subunit of mitochondrial F1.     

Solid state 31P cross-polarization/magic angle sample spinning nuclear magnetic resonance studies of crystalline glycogen phosphorylase b

³¹P cross-polarization/magic angle sample spinning nuclear magnetic resonance spectra have been obtained for pyridoxal 5′-phosphate (PLP) bound to glycogen phosphorylase b (GPb) in two different crystalline forms, monoclinic and tetragonal. Analysis of the intensities of the spinning sidebands in the nuclear magnetic resonance spectra has enabled estimates of the principal values of the ³¹P chemical shift tensors to be obtained. Differences between the two sets of values suggest differences in the environment of the phosphate moiety of the pyridoxal phosphate in the two crystalline forms. The tensor for the tetragonal crystalline form, T state GPb, is fully consistent with those found for dianionic phosphate groups in model compounds. The spectrum for the monoclinic crystalline form, R state GPb, although closer to that of dianionic than monoanionic model phosphate compounds, deviates significantly from that expected for a simple dianion or monoanion. This is likely to result from specific interactions between the PLP phosphate group and residues in its binding site in the protein. A possible explanation for the spectrum of the monoclinic crystals is that the shift tensor is averaged by a proton exchange process between different ionization states of the PLP associated with the presence of a sulfate ion bound in the vicinity of the PLP.     

Intramolecular dynamics of low molecular weight protein tyrosine phosphatase in monomer-dimer equilibrium studied by NMR: a model for changes in dynamics upon target binding

Low molecular weight protein tyrosine phosphatase (LMW-PTP) dimerizes in the phosphate-bound state in solution with a dissociation constant of K(d)=1.5(+/-0.1)mM and an off-rate on the order of 10(4)s(-1). 1H and 15N NMR chemical shifts identify the dimer interface, which is in excellent agreement with that observed in the crystal structure of the dimeric S19A mutant. Two tyrosine residues of each molecule interact with the active site of the other molecule, implying that the dimer may be taken as a model for a complex between LMW-PTP and a target protein. 15N relaxation rates for the monomeric and dimeric states were extrapolated from relaxation data acquired at four different protein concentrations. Relaxation data of satisfactory precision were extracted for the monomer, enabling model-free analyses of backbone fluctuations on pico- to nanosecond time scales. The dimer relaxation data are of lower quality due to extrapolation errors and the possible presence of higher-order oligomers at higher concentrations. A qualitative comparison of order parameters in the monomeric and apparent dimeric states shows that loops forming the dimer interface become rigidified upon dimerization. Qualitative information on monomer-dimer exchange and intramolecular conformational exchange was obtained from the concentration dependence of auto- and cross-correlated relaxation rates. The loop containing the catalytically important Asp129 fluctuates between different conformations in both the monomeric and dimeric (target bound) states. The exchange rate compares rather well with that of the catalyzed reaction step, supporting existing hypotheses that catalysis and enzyme dynamics may be coupled. The side-chain of Trp49, which is important for substrate specificity, exhibits conformational dynamics in the monomer that are largely quenched upon formation of the dimer, suggesting that binding is associated with the selection of a single side-chain conformer.     

Characterization of nucleotide binding sites on the membrane-bound chloroplast ATP synthase (coupling factor CF1)

Phosphorylation of ADP and nucleotide exchange by membrane-bound coupling factor CF₁ are very fast reactions in the light, so that a direct comparison of both reactions is difficult. By adding substrate ADP and phosphate to illuminated thylakoids together with the uncoupler FCCP, the phosphorylation time is limited and the amount of ATP formed can be reduced to less than 1 ATP per enzyme. Low concentrations of medium nucleotides during illumination increase the amount of ATP formed during uncoupling presumably by binding to the tight nucleotide binding site (further designated as ‘site A’) with an affinity of 1 to 7 μM for ADP and ATP. ATP formation itself shows half-saturation at about 30 μM. Loosely bound nucleotides are exchanged upon addition of nucleotides with uncoupler (Schumann, J. (1984) Biochim. Biophys. Acta 766, 334–342). Release depends binding of nucleotides to a second site. The affinity of this site for ADP (in the presence of phosphate) is about 30 μM. It is assumed that phosphorylation and induction of exchange both occur on the same site (site B). During ATP hydrolysis, an ATP molecule is bound to site A, while on another site, ATP is hydrolyzed rapidly. The affinity of ADP for the catalytic site (70 μM) is in the same range as the observed Michaelis constant of ADP during phosphorylation; it is assumed that site B is involved in ATP hydrolysis. Site A exhibits some catalytic activity; it might be that site A is involved in ATP formation in a dual-site mechanism. For ATP hydrolysis, however, direct determination of exchange rates showed that the exchange rate of ATP bound to site A is about 30-times lower than ATP hydrolysis under the same conditions.     

Auto-FACE: An NMR Based Binding Site Mapping Program for Fast Chemical Exchange Protein-Ligand Systems

Background

Nuclear Magnetic Resonance (NMR) spectroscopy offers a variety of experiments to study protein-ligand interactions at atomic resolution. Among these experiments, N Heteronuclear Single Quantum Correlation (HSQC) experiment is simple, less time consuming and highly informative in mapping the binding site of the ligand. The interpretation of N HSQC becomes ambiguous when the chemical shift perturbations are caused by non-specific interactions like allosteric changes and local structural rearrangement. Under such cases, detailed chemical exchange analysis based on chemical shift perturbation will assist in locating the binding site accurately.

Methodology/Principal Findings

We have automated the mapping of binding sites for fast chemical exchange systems using information obtained from N HSQC spectra of protein serially titrated with ligand of increasing concentrations. The automated program Auto-FACE (Auto-FAst Chemical Exchange analyzer) determines the parameters, e.g. rate of change of perturbation, binding equilibrium constant and magnitude of chemical shift perturbation to map the binding site residues. Interestingly, the rate of change of perturbation at lower ligand concentration is highly sensitive in differentiating the binding site residues from the non-binding site residues. To validate this program, the interaction between the protein and the ligand BH3I-1 was studied. Residues in the hydrophobic BH3 binding groove of were easily identified to be crucial for interaction with BH3I-1 from other residues that also exhibited perturbation. The geometrically averaged equilibrium constant () calculated for the residues present at the identified binding site is consistent with the values obtained by other techniques like isothermal calorimetry and fluorescence polarization assays (). Adjacent to the primary site, an additional binding site was identified which had an affinity of 3.8 times weaker than the former one. Further NMR based model fitting for individual residues suggest single site model for residues present at these binding sites and two site model for residues present between these sites. This implies that chemical shift perturbation can represent the local binding event much more accurately than the global binding event.

Conclusion/Significance

Detail NMR chemical shift perturbation analysis enabled binding site residues to be distinguished from non-binding site residues for accurate mapping of interaction site in complex fast exchange system between small molecule and protein. The methodology is automated and implemented in a program called “Auto-FACE”, which also allowed quantitative information of each interaction site and elucidation of binding mechanism.     

Tritium NMR spectroscopy of ligand binding to maltose-binding protein

Tritium-labeled alpha- and beta-maltodextrins have been used to study their complexes with maltose-binding protein (MBP), a 40-kDa bacterial protein. Five substrates, from maltose to maltohexaose, were labeled at their reducing ends and their binding studied. Tritium NMR spectroscopy of the labeled sugars showed large upfield chemical shift changes upon binding and strong anomeric specificity. At 10 degrees C, MBP bound alpha-maltose with 2.7 +/- 0.5-fold higher affinity than beta-maltose, and, for longer maltodextrins, the ratio of affinities (KD beta/KD alpha) was even larger (between 10 and 30). The maximum chemical shift change was 2.2 ppm, suggesting that the reducing end of bound alpha-maltodextrin makes close contact with an aromatic residue in the MBP-binding site. Experiments with maltotriose (and longer maltodextrins) also revealed the presence of two bound beta-maltotriose resonances in rapid exchange. We interpret these two resonances as arising from two distinct sugar-protein complexes. In one complex, the beta-maltodextrin is bound by its reducing end, and, in the other complex, the beta-maltodextrin is bound by the middle glucose residue(s). This interpretation also suggests how MBP is able to bind both linear and circular maltodextrins.