Targeting SxIP-EB1 interaction: An integrated approach to the discovery of small molecule modulators of dynamic binding sites.

End binding protein 1 (EB1) is a key element in the complex network of protein-protein interactions at microtubule (MT) growing ends, which has a fundamental role in MT polymerisation. EB1 is an important protein target as it is involved in regulating MT dynamic behaviour, and has been associated with several disease states, such as cancer and neuronal diseases. Diverse EB1 binding partners are recognised through a conserved four amino acid motif, (serine-X-isoleucine-proline) which exists within an intrinsically disordered region. Here we report the use of a multidisciplinary computational and experimental approach for the discovery of the first small molecule scaffold which targets the EB1 recruiting domain. This approach includes virtual screening (structure- and ligand-based design) and multiparameter compound selection. Subsequent studies on the selected compounds enabled the elucidation of the NMR structures of the C-terminal domain of EB1 in the free form and complexed with a small molecule. These structures show that the binding site is not preformed in solution, and ligand binding is fundamental for the binding site formation. This work is a successful demonstration of the combination of modelling and experimental methods to enable the discovery of compounds which bind to these challenging systems.

The solution structure of the complex of Lactobacillus casei dihydrofolate reductase with methotrexate.

We have determined the three-dimensional solution structure of the complex of Lactobacillus casei dihydrofolate reductase (18.3 kDa, 162 amino acid residues) formed with the anticancer drug methotrexate using 2531 distance, 361 dihedral angle and 48 hydrogen bond restraints obtained from analysis of multidimensional NMR spectra. Simulated annealing calculations produced a family of 21 structures fully consistent with the constraints. The structure has four alpha-helices and eight beta-strands with two other regions, comprising residues 11 to 14 and 126 to 127, also interacting with each other in a beta-sheet manner. The methotrexate binding site is very well defined and the structure around its glutamate moiety was improved by including restraints reflecting the previously determined specific interactions between the glutamate alpha-carboxylate group with Arg57 and the gamma-carboxylate group with His28. The overall fold of the binary complex in solution is very similar to that observed in the X-ray studies of the ternary complex of L. casei dihydrofolate reductase formed with methotrexate and NADPH (the structures of the binary and ternary complexes have a root-mean-square difference over the backbone atoms of 0.97 A). Thus no major conformational change takes place when NADPH binds to the binary complex. In the binary complex, the loop comprising residues 9 to 23 which forms part of the active site has been shown to be in the "closed" conformation as defined by M. R. Sawaya & J. Kraut, who considered the corresponding loops in crystal structures of complexes of dihydrofolate reductases from several organisms. Thus the absence of the NADPH does not result in the "occluded" form of the loop as seen in crystal studies of some other dihydrofolate reductases in the absence of coenzyme. Some regions of the structure in the binary complex which form interaction sites for NADPH are less well defined than other regions. However, in general terms, the NADPH binding site appears to be essentially pre-formed in the binary complex. This may contribute to the tighter binding of coenzyme in the presence of methotrexate.

The conformation of coenzyme A bound to chloramphenicol acetyltransferase determined by transferred NOE experiments.

The conformation of coenzyme A bound to chloramphenicol acetyltransferase has been studied in solution by NMR methods. Transferred nuclear Overhauser enhancement (NOE) and rotating frame NOE (ROE) experiments were used to determine the conformation of the bound coenzyme. Experiments were carried out at five mixing times and two temperatures, and with normal and perdeuterated enzyme, to ensure (1) that the fast exchange condition was satisfied and (2) that the results were not complicated by spin diffusion involving enzyme protons. The data were analysed using a general approach involving combined exchange and relaxation matrices. For the binary complex of coenzyme A (CoA) and enzyme, the conformation of CoA was calculated by using distance constraints derived from the intensities of 71 NOE and 33 ROE cross-peaks between coenzyme protons. The conformation of the adenosine moiety of CoA in the structure deduced by NMR is very close to that seen in the crystal structure of this complex, while the pantetheine moiety is clearly less extended. Essentially the same conformation was obtained whether or not the calculations included the protein (with appropriate intermolecular energy terms). The difference between the NMR and X-ray structures is interpreted in terms of the existence of two conformations of the CoA-enzyme complex. Support for this model comes from measurements of the coenzyme dissociation rate constant; NMR (lineshape analysis and transferred NOE experiments) gives estimates of koff approximately 3700 s-1 at 298 K and approximately 500 s-1 at 280 K, both significantly greater than estimates by fluorescence stopped-flow measurements. For the ternary complex of CoA, chloramphenicol and enzyme, 71 NOE cross peaks between protons of coenzyme A and a further ten cross-peaks between protons of coenzyme A and chloramphenicol were measured. Starting with a model derived from the crystal structures of the two binary complexes (in the absence of crystallographic data for the ternary complex) the conformations and relative positions of the two ligands were refined using the distance constraints derived from these NOEs. The conformation of the adenosine part of CoA is the same as in the binary complex, while the pantetheine arm is more extended and approaches close to the bound chloramphenicol molecule. The model of the ternary complex is discussed in terms of the information available on the mechanism of the enzyme.

Role of the active-site carboxylate in dihydrofolate reductase: kinetic and spectroscopic studies of the aspartate 26-->asparagine mutant of the Lactobacillus casei enzyme.

A mutant of Lactobacillus casei dihydrofolate reductase, D26N, in which the active site aspartic acid residue has been replaced by asparagine by oligonucleotide-directed mutagenesis has been studied by NMR and optical spectroscopy and its kinetic behavior characterized in detail. On the basis of comparisons of a large number of chemical shifts and NOEs, it is clear that there are only very slight structural differences between the methotrexate complexes of the wild-type and mutant enzymes and that these are restricted to the immediate environment of the substitution. The data suggest a slight difference in orientation of the pteridine ring in the binding site in the mutant enzyme. Both NMR and UV spectroscopy show that methotrexate is protonated on N1 when bound to the wild-type enzyme but not when bound to the mutant. Binding constant measurements by fluorescence quenching and steady-state kinetic measurements of dihydrofolate (FH2) and folate reduction show that the substitution has little or no effect on substrate, coenzyme, and inhibitor binding (< 7-fold increase in Kd) and only a modest effect on kcat (up to a factor of 9 for FH2 and 25 for folate) and kcat/KM (up to a factor of 13 for FH2 and 14 for folate). Measurements of deuterium isotope effects and direct measurements of hydride ion transfer and product release by stopped-flow methods revealed that for the mutant enzyme hydride ion transfer is rate-limiting across the pH range 5-8. This allowed a direct comparison of the rate of hydride ion transfer in the wild-type and mutant enzymes; the asparagine substitution was found to decrease this rate by 62-fold at pH 5.5 and 9-fold at pH 7.5. This effect is much smaller than that seen for the corresponding mutant of Escherichia coli dihydrofolate reductase [Howell, E. E., Villafranca, J. E., Warren, M. S., Oatley, S. J., & Kraut, J. (1986) Science 231, 1123-1128], estimated as a 1000-fold decrease in the rate of hydride ion transfer. The change in pH dependence of kcat resulting from the substitution is consistent with, but does not prove, the idea that the group of pK 6.0 which must be protonated for hydride ion transfer to occur is Asp26. For folate reduction, the pH dependence of kcat is determined by two pKs, one of which, pK 5, disappears in the mutant enzyme, suggesting that it may correspond to ionization of Asp26.(ABSTRACT TRUNCATED AT 400 WORDS)

Model for the complex between protein G and an antibody Fc fragment in solution.

BACKGROUND: Streptococcal protein G and staphylococcal protein A are bacterial antibody-binding proteins, widely used as immunological tools, whose antibody-binding domains are structurally quite different. The binding of protein G to Fc fragments is competitive with respect to protein A, suggesting that the binding sites for protein A and protein G on Fc overlap, notwithstanding the fact that they lack sequence or structural similarity. RESULTS: To resolve this issue, the residues involved in the interaction between an IgG-binding domain of protein G (domain II) and the Fc fragment of mouse IgG2a have been identified by use of 13C and 15N NMR. Binding of protein G domain II selectively perturbed resonances from residues between the CH2 and CH3 domains of Fc, whereas in domain II the residues affected are primarily those on the alpha-helix and the third strand of the beta-sheet. This information was used, together with the structures of the two uncomplexed proteins, to construct a model of the complex, using Monte Carlo minimization techniques. In this model, the alpha-helix of protein G lies in the same position as helix 1 of protein A in the crystal structure of the protein A:Fc complex, but its orientation differs from the latter by 180 degrees. CONCLUSIONS: The interactions of the bacterial antibody-binding proteins with their 'target' immunoglobulins involve a very versatile set of protein-protein interactions. First, the IgG-binding domains of protein A and protein G have quite different three-dimensional structures, but bind to sites on the Fc fragment that overlap extensively. Secondly, protein G employs two quite different regions of its surface to bind to the Fab and Fc regions of IgG.

Three-dimensional structure of proteolytic fragment 163-231 of bacterioopsin determined from nuclear magnetic resonance data in solution.

546 NOESY cross-peak volumes were measured in the two-dimensional NOESY spectrum of proteolytic fragment 163-231 of bacterioopsin in organic solution. These data and 42 detected hydrogen bonds were applied for determining the peptide spatial structure. The fold of the polypeptide chain was determined by local structure analysis, a distance geometry approach and systematic search for energetically allowed side-chain rotamers which are consistent with experimental NOESY cross-peak volumes. The effective rotational correlation time of 6 ns for the molecule was evaluated from optimization of the local structure to meet NOE data and from the dependence on mixing time of the NiH/Ci alpha H cross-peak volumes of the residues in alpha-helical conformation. The resulting structure has two well defined alpha-helical regions, 168-191 and 198-227, with root-mean-square deviation 44 pm and 69 pm, respectively, between the backbone atoms in 14 final energy refined conformations. The alpha-helices correspond to transmembrane segments F and G of bacteriorhodopsin. The segment F contains proline 186, which introduces a kink of about 25 degrees with a disruption of the hydrogen bond with the NH group of the following residue. The segments are connected by a flexible loop region 192-197. Torsion angles chi 1 are unequivocally defined for 62% of side chains in the alpha-helices but half of them differ from electron cryo-microscopy (ECM) model of bacteriorhodopsin, apparently because of the low resolution of ECM. Nevertheless, the F and G segments can be packed as in the ECM model and with side-chain conformations consistent with all NMR data in solution.

The divalent cation-binding sites of gramicidin A transmembrane ion-channel.

The conductance of the gramicidin A single channels in glycerolmonooleate membranes is strongly reduced in the presence of Mn2+ cations. The nmr experiments were performed for N-terminal to N-terminal gramicidin A dimer formed by two right-handed single-stranded helixes incorporated into the sodium dodecyl sulfate micelles in the presence of Mn2+ ions. Dependence of the nonselective spin-lattice relaxation rates of the gramicidin A protons on Mn2+ concentration was analyzed to determine coordinates of the divalent cation binding sites. It is inferred that Mn2+ ions are bound at the channel mouths at distances of 6.4, 8.6, and 8.8 A (+/- 2 A) from the oxygen atoms of exposed carbonyl groups of D-Leu 12, 14, and 10, respectively. The bounded Mn2+ retains its hydrate shell, the size of which (approximately 6 A) exceeds the inner pore diameter (approximately 4 A). That makes the gramicidin A channel impermeable for divalent cations.

Sequence-specific 1H-NMR assignment and conformation of proteolytic fragment 163-231 of bacterioopsin.

Proteolytic fragment 163-231 of bacterioopsin was isolated from Halobacterium halobium purple membrane treated with NaBH4 and papain under nondenaturing conditions. Two-dimensional 1H-NMR spectra of (163-231)-bacterioopsin solubilized in chloroform/methanol (1:1), 0.1 M LiClO4 indicated the existence of one predominant conformation. Most of the resonances in the 1H-NMR spectra of (163-231)-bacterioopsin were assigned by two-dimensional techniques. Two extended right-handed alpha-helical regions Ala168-Ile191 and Asn202-Arg227 were identified on the basis of NOE connectivities and deuterium exchange rates. The N-terminal part of the peptide is flexible and the region of Gly192-Leu201 adopts a specific conformation. The protons of OH groups of Thr178, Ser183 and Ser214 slowly exchange with solvent, and side-chain conformations of these residues, as evaluated by NOE connectivities of OH protons, are optimal for the formation of hydrogen bonds between OH and backbone carbonyl groups.

[Spatial structure of gramicidin A in organic solvents. 1H-NMR analysis of conformation heterogeneity in ethanol]. / Prostranstvennye struktury gramitsidina A v organicheskikh rastvoriteliakh. 1H-IaMR-analiz konformatsionnoi geterogennosti v étanole.

Structural features of double helices formed by polypeptides with alternating L- and D-amino acid residues were analysed. It was found that the map of short distances (less than 4 A) between protons of the two backbones is unique for each double helix type and even its fragment implies unambiguously parameters of the helix (i.e. parallel or antiparallel, handedness, pitch of helix, relative shift of polypeptide chains). By analysis of two-dimensional 1H-NMR spectra (COSY, RELSY, HOHAHA, NOESY), proton resonances of [Val1]gramicidin A (GA) in the ethanol solution were assigned. The results obtained show that the solution contains five stable conformations of GA in comparable concentrations. Monomer of GA is in a random coil conformation. Specific maps of short interproton distances for the other four species (1-4) were obtained by means of two dimensional nuclear Overhauser effect spectroscopy. The maps as well as spin-spin couplings of the H-NC alpha-H protons and solvent accessibilities of the individual amide groups correspond to four types of double helices pi pi LD 5,6 with 5.6 residues per turn. The double helices are related to the Veatch species 1-4 of GA. Species 1 and 2 are left-handed parallel double helices increase increase pi pi LD 5,6 with different relative shift of polypeptide chains. Species 3 is a left-handed antiparallel double helix increase decrease pi pi LD 5,6 and species 4 is a right-handed parallel double helix increase increase LD 5,6. In the dimers helices are fixed by the maximum number (28) of interbackbone hydrogen bonds NH...O = C possible for these structures. Species 1, 3 and 4 have C2 symmetry axes. Relationship between gramicidin A spatial structures induced by various media is discussed.

1H-NMR study of gramicidin A transmembrane ion channel. Head-to-head right-handed, single-stranded helices.

The structure of [Val1]gramicidin A incorporated into sodium dodecyl-d25 sulphate micelles has been studied by two-dimensional proton NMR spectroscopy. Analysis of nuclear Overhauser effects, spin-spin couplings and solvent accessibility of NH groups show that the conformation of the Na+ complex of gramicidin A in detergent micelles, which in many ways mimic the phospholipid bilayer of biomembranes, is an N-terminal to N-terminal (head-to-head) dimer (Formula: see text) formed by two right-handed, single-stranded beta 6.3 helices with 6.3 residues per turn, differing from Urry's structure by handedness of the helices.