Solution structure of the C-terminal SH2 domain of the human tyrosine kinase Syk complexed with a phosphotyrosine pentapeptide.

BACKGROUND: Recruitment of the intracellular tyrosine kinase Syk to activated immune-response receptors is a critical early step in intracellular signaling. In mast cells, Syk specifically associates with doubly phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs) that are found within the IgE receptor. The mechanism by which Syk recognizes these motifs is not fully understood. Both Syk SH2 (Src homology 2) domains are required for high-affinity binding to these motifs, but the C-terminal SH2 domain (Syk-C) can function independently and can bind, in isolation, to the tyrosine-phosphorylated IgE receptor in vitro. In order to improve understanding of the cellular function of Syk, we have determined the solution structure of Syk-C complexed with a phosphotyrosine peptide derived from the gamma subunit of the IgE receptor. RESULTS: The Syk-C:peptide structure is compared with liganded structures of both the SH2 domain of Src and the C-terminal SH2 domain of ZAP-70 (the 70 kDa zeta-associated protein). The topologies of these domains are similar, although significant differences occur in the loop regions. In the Syk-C structure, the phosphotyrosine and leucine residues of the peptide ligand interact with pockets on the protein, and the intervening residues are extended. CONCLUSIONS: Syk-C resembles other SH2 domains in its peptide-binding interactions and overall topology, a result that is consistent with its ability to function as an independent SH2 domain in vitro. This result suggests that Syk-C plays a unique role in the intact Syk protein. The determinants of the binding affinity and selectivity of Syk-C may reside in the least-conserved structural elements that comprise the phosphotyrosine- and leucine-binding sites. These structural features can be exploited for the design of Syk-selective SH2 antagonists for the treatment of allergic disorders and asthma.

Amide proton hydrogen exchange rates for sperm whale myoglobin obtained from 15N-1H NMR spectra.

The hydrogen exchange behavior of exchangeable protons in proteins can provide important information for understanding the principles of protein structure and function. The positions and exchange rates of the slowly-exchanging amide protons in sperm whale myoglobin have been mapped using 15N-1H NMR spectroscopy. The slowest-exchanging amide protons are those that are hydrogen bonded in the longest helices, including members of the B, E, and H helices. Significant protection factors were observed also in the A, C, and G helices, and for a few residues in the D and F helices. Knowledge of the identity of slowly-exchanging amide protons forms the basis for the extensive quench-flow kinetic folding experiments that have been performed for myoglobin, and gives insights into the tertiary interactions and dynamics in the protein.

3D solution structure of copper and silver-substituted yeast metallothioneins.

3D solution structural calculations for yeast silver(I)-substituted metallothionein (MT) and native copper(I) MT were completed using experimentally determined NOE and dihedral angle constraints, in conjunction with experimentally derived metal-to-Cys connectivities for AgMT which were assumed identical for CuMT. For the first 40 residues in both structures, the polypeptide backbone wraps around the metal cluster in two large parallel loops separated by a deep cleft containing the metal cluster. Minor differences between the two structures include differences in hydrogen bonds and the orientation of the N-terminus with the overall protein volume conserved to within 6.5%.

Intramolecular conformation of puromycin in solution as studied by proton magnetic resonance.

The intramolecular conformation of puromycin, a broad spectrum antibiotic, in solution has been investigated by proton magnetic resonance (PMR) spectroscopy. A comparison of the proton chemical shift and proton-proton coupling constant data of puromycin with puromycin aminonucleoside suggests that puromycin in solution exists as an equilibrium blend of extended and folded conformers. These folded conformers are the result of flexibility around the C alpha -C beta bond of the aminoacyl segment of puromycin. One of the folded conformers predicted by PMR is in excellent agreement with the x-ray data.

Self-association of puromycin as studied by proton magnetic resonance spectroscopy.

The self-association of puromycin has been studied using proton magnetic resonance spectroscopy. The concentration, temperature and pH dependence studies of the proton chemical shifts of the adenine protons indicate that puromycin in aqueous solution at pD 7.4 self associates predominantly through adenine-adenine interaction. At this pD, the amino group of the aminoacyl segment of puromycin has been demonstrated to exist in a equilibrium blend of protonated and non-protonated forms. At pD 2.6, PM is found to exist predominantly in the monomeric from in which the methyl groups of the 6N-dimethyladenine are found to be non-equivalent due to hindered rotation about the C6-N6 bond.

Copper- and silver-substituted yeast metallothioneins: sequential 1H NMR assignments reflecting conformational heterogeneity at the C terminus.

Complete 1H NMR sequential assignments have been made for copper(I)- and silver (I)-substituted metallothionein (MT) from Saccharomyces cerevisiae using standard 2D 1H NMR methods. The fingerprint region of the COSY spectrum of both metalloproteins shows a doubling of a few backbone proton resonances from residue K41 onward in the C terminus. This doubling of resonances is absent in the spectrum of the truncated mutant protein that lacks the five C-terminal residues which includes two cysteines. Concurrently, it has been established from a comparison of the heteronuclear 1H-109 Ag multiple-quantum coherence transfer (HMQC) spectrum on the silver-substituted mutant and the wild-type protein that metal ligation is similar in both molecules. Thus, the 2 C-terminal Cys are not essential for metal cluster formation in the wild-type yeast MT and only 10 of the 12 Cys present in this protein appear to be involved in ligating the 7 mol of bound metal ions. A qualitative analysis of the coupling constant, hydrogen exchange, and NOE data indicates the presence of many type I beta-turns and the lack of any other regular secondary structural elements. A comparison of chemical shifts and NOE data for native copper- and silver-substituted yeast MT indicates a high degree of conservation of structural elements in both proteins. Therefore, it seems reasonable to conclude that the metal to Cys connectivities which are obtained directly from the HMQC data on silver-substituted metallothionein are conserved in the native copper protein. Interestingly, a mixture of both 2 and 3 coordination was found for the bound Ag(I) ions in a single Ag7Cys10 cluster. This mixed coordination number and a single cluster arrangement is most probably also shared with the Cu(I) ion coordination in the native protein.

Three-dimensional solution structure of Callinectes sapidus metallothionein-1 determined by homonuclear and heteronuclear magnetic resonance spectroscopy.

Metallothionein is a cysteine-rich metal-binding protein whose biosynthesis is closely regulated by the level of exposure of an organism to zinc, copper, cadmium, and other metal salts. The metallothionein from Callinectes sapidus is known to bind six divalent metal ions in two separate metal-binding clusters. Heteronuclear 1H-113Cd and homonuclear 1H-1H NMR correlation experiments have been used to establish that the two clusters reside in two distinct protein domains. The three-dimensional solution structure of the metallothionein has been determined using the distance and angle constraints derived from these two-dimensional NMR data sets and a distance geometry/simulated annealing protocol. There are no interdomain short distance (< or = 4.5 A) constraints observed in this protein, enabling the calculation of structures for the N-terminal, beta domain and the C-terminal, alpha domain separately. A total of 18 structures were obtained for each domain. The structures are based on a total of 364 experimental NMR restraints consisting of 277 approximate interproton distance restraints, 12 chi 1 and 51 phi angular restraints, and 24 metal-to-cysteine connectivities obtained from 1H-113Cd correlation experiments. The only element of regular secondary structure in either of the two domains is a short segment of helix in the C-terminal alpha domain between Lys42 and Thr48. The folding of the polypeptide backbone chain in each domain, however, gives rise to several type I beta turns. There are no type II beta turns.

NMR studies of the heme pocket conformations of monomeric hemoglobins from Glycera dibranchiata. Implications for ligand binding.

Two-dimensional 1H-NMR methods have been used to assign side-chain resonances for the tryptophan residues and for several amino acids located in the heme pockets of the carbon monoxide complexes of the major monomeric hemoglobins from Glycera dibranchiata. The NMR spectra reveal a high degree of conservation of the heme pocket structure in the different hemoglobins. However some conformational differences are evident and residues at positions B10 and G8 on the distal side of the heme pocket are not conserved. From the present NMR studies it appears that the monomeric G. dibranchiata hemoglobin examined by X-ray crystallography [Padlan, E. A. & Love, W. (1974) J. Biol. Chem. 249, 4067-4078] corresponds to HbC. Except that the orientation of the heme in solution is the reverse of that reported in the crystal structure, there is a close correspondence between the heme pocket structure in the crystal and in solution. The proximal histidine coordination geometry is almost identical in the CO complexes of the three monomeric hemoglobins studied. Distal residues are strongly implicated in determining the observed kinetic differences in ligand binding reactions. In particular, steric crowding of the ligand binding site in hemoglobin A is probably a major factor in the slower kinetics of this component.

NMR studies of the conformations of leghemoglobins from soybean and lupin.

Phase-sensitive two-dimensional NMR methods have been used to obtain extensive proton resonance assignments for the carbon monoxide complexes of lupin leghemoglobins I and II and soybean leghemoglobin a. The assigned resonances provide information on the solution conformations of the proteins, particularly in the vicinity of the heme. The structure of the CO complex of lupin leghemoglobin II in solution is compared with the X-ray crystal structure of the cyanide complex by comparison of observed and calculated ring current shifts. The structures are generally very similar but significant differences are observed for the ligand contact residues, Phe30, His63 and Val67, and for the proximal His97 ligand. Certain residues are disordered and adopt two interconverting conformations in lupin leghemoglobin II in solution. The proximal heme pocket structure is closely conserved in the lupin leghemoglobins I and II but small differences in conformation in the distal heme pocket are apparent. Larger conformational differences are observed when comparisons are made with the CO complex of soybean leghemoglobin. Altered protein-heme packing is indicated on the proximal side of the heme and some conformational differences are evident in the distal heme pocket. The small conformational differences between the three leghemoglobins probably contribute to the known differences in their O2 and CO association and dissociation kinetics. The heme pocket conformations of the three leghemoglobins are more closely related to each other than to sperm whale myoglobin. The most notable differences between the leghemoglobins and myoglobin are: (a) reduced steric crowding of the ligand binding site in the leghemoglobins, (b) different orientations of the distal histidine, and (c) small but significant differences in proximal histidine coordination geometry. These changes probably contribute to the large differences in ligand binding kinetics between the leghemoglobins and myoglobin.

Identification of transient intermediates in the bisphosphatase reaction of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase by 31P-NMR spectroscopy.

31P-NMR spectroscopy was used to identify reaction intermediates during catalytic turn-over of the fructose-2,6-bisphosphatase domain (Fru-2,6-P2ase) of the bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. When fructose-2,6-bisphosphate (Fru-2,6-P2) was added to the enzyme, the 31P-NMR spectrum showed three resonances in addition to those of free substrate: the phosphohistidine (His-P) intermediate, the C-6 phosphoryl group of fructose-6-phosphate bound to the phosphoenzyme, and phosphate generated by the hydrolysis of substrate. Direct analysis of the alkali-denatured phospho-enzyme intermediate by 1H-31P heteronuclear multiple quantum-filtered coherence spectroscopy confirmed the formation of 3-N-phosphohistidine. Binding of fructose 6-phosphate to the bisphosphatase was detected by a down-field shift and broadening of the C-6 phosphoryl resonance. The down-field shift was greater in the presence of the phosphoenzyme intermediate. Inhibition of Fru-2,6-P2 hydrolysis by fructose 6-phosphate and Fru-2,6-P2 was shown to involve binding of the sugar phosphates to the phosphoenzyme. This study provides new experimental evidence in support of the reaction mechanism of Fru-2,6-P2ase and suggests that the steady-state His-P intermediate exists primarily in the E-P.fructose 6-phosphate complex. These results lay a solid foundation for the use of 31P-NMR magnetization transfer studies to provide an in-depth analysis of the bisphosphatase reaction mechanism.

Mutation of invariant cysteines of mammalian metallothionein alters metal binding capacity, cadmium resistance, and 113Cd NMR spectrum.

Using a yeast expression vector system, we have expressed both wild type and six mutated Chinese hamster metallothionein coding sequences in a metal-sensitive yeast strain in which the endogenous metallothionein gene has been deleted. The mutant proteins have single or double cysteine to tyrosine replacements (C13Y, C50Y, and C13,50Y), single cysteine to serine replacements (C13S and C50S), or a single cysteine to alanine replacement (C50A). These proteins function in their yeast host in cadmium detoxification to differing extents. Metallothioneins which contain a cysteine mutation at position 50 (C50Y, C50S, C50A, and C13,50Y) conferred markedly less cadmium resistance than wild type metallothionein, or metallothionein with a single cysteine mutation at position 13 (C13Y and C13S). Wild type and three of the mutant Chinese hamster metallothioneins (C13Y, C50Y, and C13,50Y) were purified from yeast grown in subtoxic levels of either CdCl2 or 113CdCl2. All three of the mutant proteins bound less cadmium than the wild type protein when metal-binding stoichiometries were determined. The one-dimensional 113Cd NMR spectrum of the recombinant wild type Chinese hamster metallothionein was compared to the spectra of native rat and rabbit liver metallothioneins. The close correspondence between the 113Cd chemical shifts in these metallothioneins is consistent with the presence of two separate metal clusters, A and B, corresponding, respectively, to the alpha- and beta-domains, in the recombinant metallothionein. The one-dimensional 113Cd NMR spectra recorded on each of the three mutant metallothioneins, on the other hand, provide some indication as to the structural basis for the reduced, by one, metal stoichiometry of each of the mutant metallothioneins. For the C13Y mutant, it appears that the beta-domain now binds a total of two metal ions whereas with the C50Y mutant, the alpha-domain appears metal-deficient. For the double mutant, C13,50Y, the 113Cd resonances are indicative of major structural reorganizations in both domains.

Structure-based design and synthesis of a novel class of Src SH2 inhibitors.

The structure-based design and synthesis of a novel class of 2,4-disubstituted thiazoles as Src SH2 inhibitors is described. Initial results are presented, including the X-ray and NMR analysis of one thiazole inhibitor bound to Lck and Src SH2.

An efficient synthesis of a 4'-phosphonodifluoromethyl-3'-formyl-phenylalanine containing SRC SH2 ligand.

A CuBr-mediated, regioselective cross-coupling between methyl 2,5-diiodobenzoate (4) and [(diethoxyphosphinyl)difluoromethyl]zinc bromide is reported. Palladium-catalyzed incorporation of an amino acid side chain, followed by subsequent modifications resulted in the rapid construction of 2. Compound 2 was designed to engage Cys188 of the Src SH2 domain, however, this was not observed spectroscopically.

Selective inhibition of Src SH2 by a novel thiol-targeting tricarbonyl-modified inhibitor and mechanistic analysis by (1)H/(13)C NMR spectroscopy.

Detailed analysis of Src SH2 binding by peptides containing a novel tricarbonyl-modified pTyr moiety is described. We envisaged that Src SH2 selectivity might be obtained by exploiting the thiol group of Cys188 present in the pTyr binding pocket of the protein at the betaC3 position. Peptidyl as well as non-peptidyl compounds 1-4 possessing a 4-alpha,beta-diketoester-modified pTyr mimic exhibited micromolar affinity to Src SH2. Furthermore, these tricarbonyl compounds were selective for Src SH2 to the extent they showed no significant affinity for either Cys188Ser or Cys188Ala Src SH2 mutants. Upon closer examination of the binding of these tricarbonyls to Src SH2 using NMR of 13C-labeled compounds (6a, 6b, and 6c), we found that after the initial binding event the molecule disproportionated in a 'retro-Claisen' fashion to provide benzoic acid 16 and, following hydrolysis of the methyl ester 17, the hemiketal adduct of glyoxalic acid 18.