An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.
Src homology (SH)2 and SH3 domains are found in a variety of proteins involved in the control of cellular signaling and architecture. The possible interrelationships between domains are not easily investigated, even though several cases of multiple domain-containing constructs have been studied structurally. As a complement to direct structural methods, we have developed consolidated ligands and tested their binding to the Abl SH(32) complex. Consolidated ligands combine in the same molecule peptide sequences recognized by SH2 and SH3 domains, i.e., Pro-Val-pTyr-Glu-Asn-Val and Pro-Pro-Ala-Tyr-Pro-Pro-Pro-Pro-Val-Pro, respectively; these are joined by oligoglycyl linkers. Four types of ligands were chemically synthesized, representing all the possible relative orientations of ligands. Their affinities were found to vary with binding portion topologies and linker lengths. Two of these types were shown to bind to both SH2 and SH3 dual domains with high affinities and specificities, showing increases of one order of magnitude, as compared to the most strongly bound monovalent equivalent. These results suggest that the relative orientation of SH2 and SH3 in Abl SH(32) is not fixed, and this synthetic approach may be generally useful for determining the structures of ligated complexes and for developing reagents with high affinities and specificities.
The Per-ARNT-Sim (PAS) domain serine/threonine kinase PAS kinase is involved in energy flux and protein synthesis. In yeast, PSK1 and PSK2 are two partially redundant PASK homologs. We recently generated PSK2 deletion mutant and showed that Psk2 acts as a nutrient-sensing protein kinase to modulate Ultradian clock-coupled respiratory oscillation in yeast. Here, we show that deletion of PSK1 increased the sensitivity of yeast cells to oxidative stress (H2 O2 treatment) and partially inhibited cell growth; however, the growth of the PSK2-deleted mutant was similar to that of the wild type. Superoxide dismutase-1 (SOD1) mRNA and protein levels were lower in PSK1-deletion mutant than the wild type. The mRNA levels of stress response genes CTT1, HSP104, ATH1, NTH1 and SOD2 were similar in both the PSK1-deleted mutant and wild-type yeast. Furthermore, intracellular accumulation of reactive oxygen species (ROS) was noted in PSK1-deleted mutant. These results suggest that PSK1 induces SOD1 expression to protect against oxidative stress in yeast.
The derivative N alpha-9-fluorenylmethyloxycarbonyl-O-phospho-L-tyrosine [Fmoc-Tyr(PO3H2)-OH] has been used successfully for the solid-phase synthesis of a wide variety of phosphorylated peptides. However, when it is used to incorporate consecutive phosphotyrosine residues, a pyrophosphate linkage can form between the two adjacent tyrosines. Incorporation of unprotected phosphotyrosine during the synthesis of peptides with multiple phosphotyrosine residues has been studied as a function of coupling conditions and the absence or presence of intervening amino acid residues. The pyrophosphate-forming side reaction is more severe with increased coupling times and/or repetitions of coupling and occurs only when the phosphotyrosine residues are directly adjacent to one another.
Twenty peptides containing hydroxy-amino acids have been synthesized manually by stepwise solid-phase procedure. The chloromethyl resin and MBHA resin were used as solid supports. A new reagent of 0.5 mol.L-1 DDSi/1.5 mol.L-1 phenol/DCM was applied for the removal of N alpha-Boc group. TFMSA was the cleaving reagent. After purification by C-18 column; all products were assayed according to amino acid analysis. The bioactivity of synthetic peptides was tested for the effect on progesterone production in vitro. Eight peptides, GlyTyrAlaLys, (SarSer)2Lys and its methyl ester, TyrLys, HisTyr-NH2, ThrProTyrLys-NH2 TyrThrProArgLys and AspHisProThrPheLys showed significant effect on inhibiting hCG-induced progesterone production, and first three of them could also inhibit basal progesterone secretion. However, peptide GlySerTyr exhibited stimulative activity on basal progesterone secretion. So far, no reasonable relationship between structure and bioactivity was found.
An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.
An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.
The possible interrelationships between multiple domains of proteins involved in intracellular signal transduction are complex and not easily investigated. We have synthesized a series of bivalent consolidated ligands, which interact simultaneously with the SH2 and SH3 domain of Abelson kinase in a SH(32) dual domain construct, a portion of native Abelson kinase. Affinities were measured by quenching of intrinsic tryptophan fluorescence. Consolidated ligands have enhanced affinity and specificity compared to monovalent equivalents. Affinity is also dependent on the length of the linker joining the two parts, with an optimum distance similar to that expected from structural models of Abl (SH(32). These results suggest that consolidated ligands may be generally useful reagents for probing structural and functional activities of multidomain proteins. The possible interrelationships between multiple domains of proteins involved in intracellular signal transduction are complex and not easily investigated. We have synthesized a series of bivalent consolidated ligands, which interact simultaneously with the SH2 and SH3 domain of Abelson kinase in a SH(32) dual domain construct, a portion of native Abelson kinase. Affinities were measured by quenching of intrinsic tryptophan fluorescence. Consolidated ligands have enhanced affinity and specificity compared to monovalent equivalents. Affinity is also dependent on the length of the linker joining the two parts, with an optimum distance similar to that expected from structural models of Abl (SH(32). These results suggest that consolidated ligands may be generally useful reagents for probing structural and functional activities of multidomain proteins. Src homology (SH) 1The abbreviations used are: SHSrc homologyFmocN-(9-fluorenyl)methoxycarbonylDMFN,N-dimethylformamideDde1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethylBoct-butoxycarbonylBOPbenzotriazol-1-yloxytris (dimethylamino) phosphoniumHOBt1-hydroxybenzotriazoleNMMN-methylmorpholineDIEAN,N-diisopropylethylaminePEG-PSpolyethylene glycol-polystyrene graft supportPALpeptide amide linker, i.e. 5-(4-(9-fluorenylmethyloxycarbonyl)aminomethyl-3,5-dimethoxyphenoxy)valeric acid. domains are building blocks in many proteins involved in intracellular signal transduction. Detailed understanding of the pathways involving these domains is complicated by the substantial range of individual specificities in SH2 and SH3 domains, and their combination into large proteins that can form multiple homo- and heteromolecular associations. The reductionist approach of studying individual domains has been very successful(1Kuriyan J. Cowburn D. Curr. Opin. Struct. Biol. 1993; 3: 828-837Crossref Scopus (107) Google Scholar, 2Pawson T. Nature. 1995; 373: 573-580Crossref PubMed Scopus (2234) Google Scholar, 3Cohen G.B. Ren R. Baltimore D. Cell. 1995; 80: 237-248Abstract Full Text PDF PubMed Scopus (926) Google Scholar). Nonetheless, the interactions between the domains are still poorly understood. These interactions are likely to be of significance in explaining more fully the complete activities of the signal-transducing complexes. A detailed structural picture of the inter- and intramolecular organization of the domains is then a significant objective. Several cases of multiple SH2 and SH3 domain-containing constructs have been studied structurally (Lck SH(32) ((4Eck M.J. Atwell S.K. Shoelson S.E. Harrison S.C. Nature. 1994; 368: 764-769Crossref PubMed Scopus (240) Google Scholar), Grb2 SH(323) (5Maignan S. Guilloteau J.-P. Becquart J. Ducruix A. Science. 1995; 268: 291-293Crossref PubMed Scopus (181) Google Scholar), and Abl SH(32) (6Gosser Y.Q. Zheng J. Overduin M. Mayer B.J. Cowburn D. Structure. 1995; 3: 1075-1088Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar)), but there are technical limitations to current structural approaches. In the crystalline state, packing forces may be of the same magnitude as the weak interdomain forces, so there are limits to the interpretation of diffraction studies. Solution studies by NMR are only applicable in a molecular mass range less than ~30 kDa. For NMR, time-averaged NOE constraints in rapidly exchanging conformations are not readily interpretable. As a complement to direct structural methods, we propose the investigation of such multidomain complexes using "consolidated" ligands. These ligands, having multiple binding portions, may be expected to bind with high affinity and specificity when a linker between the two affine segments is of the correct length, and there is little affinity of the linker itself. The consolidated ligands do not necessarily resemble any natural ligand. Such ligands are demonstrated here, binding to the SH(32) of Abelson kinase. It is reasonable to assume that consolidated ligands for dual SH2, dual SH3, or multiple combinations with other ligands can be produced using a similar approach. Src homology N-(9-fluorenyl)methoxycarbonyl N,N-dimethylformamide 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl t-butoxycarbonyl benzotriazol-1-yloxytris (dimethylamino) phosphonium 1-hydroxybenzotriazole N-methylmorpholine N,N-diisopropylethylamine polyethylene glycol-polystyrene graft support peptide amide linker, i.e. 5-(4-(9-fluorenylmethyloxycarbonyl)aminomethyl-3,5-dimethoxyphenoxy)valeric acid. The proposed consolidated ligands are similar in concept to affinity reagents, with the modification that the second functionality is a binding element rather than a reactive moiety. Consolidated ligands may also be useful reagents for studies of the cell biology of the signal transduction complexes (for example, in additional combination with antibodies or reporter groups like fluorescent tags), and may provide leads into possible classes of diagnostic and therapeutic agents in the many areas of pathology in which SH domains are involved. This approach will be useful where micromolar affinities of ligands to single SH domains provide insufficient affinity, and hence specificity, for pharmacological action (e.g.(7Gibbs J.B. Oliff A. Kohl N.E. Cell. 1994; 77: 175-178Abstract Full Text PDF PubMed Scopus (510) Google Scholar) and (8Levitzki A. Gazit A. Science. 1995; 267: 1782-1788Crossref PubMed Scopus (1628) Google Scholar)). The protein target for this study is the regulatory apparatus, SH(32) (4Eck M.J. Atwell S.K. Shoelson S.E. Harrison S.C. Nature. 1994; 368: 764-769Crossref PubMed Scopus (240) Google Scholar), of human Abelson kinase (Abl). Abl was originally isolated as the gene product from abl of murine leukemia virus; the human abl gene has been isolated and shown, in cases of chronic myologenous leukemia, to be a causative factor in a fusion following chromosomal translocation (reviewed in (9Mayer B.J. Hirai H. Sakai R. Curr. Biol. 1995; 5: 296-305Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar)). Ligands of moderate affinity have been identified for the isolated SH2 (10Zhu G. Decker S.J. Maclean D. McNamara D.J. Singh J. Sawyer T.K. Saltiel A.R. Oncogene. 1994; 9: 1379-1385PubMed Google Scholar) and SH3 domains (11Cicchetti P. Mayer B.J. Thiel G. Baltimore D. Science. 1992; 257: 803-806Crossref PubMed Scopus (420) Google Scholar, 12Ren R. Mayer B.K. Cicchetti P. Baltimore D. Science. 1993; 259: 1157-1161Crossref PubMed Scopus (1022) Google Scholar). For Abl SH3, a crystal structure of the complex of the ligand 3BP-1 is available(13Musacchio A. Saraste M. Wilmanns M. Nature Struct. Biol. 1994; 1: 546-551Crossref PubMed Scopus (283) Google Scholar), indicating that the ligand is in the so-called class I orientation (reviewed in (2Pawson T. Nature. 1995; 373: 573-580Crossref PubMed Scopus (2234) Google Scholar)). Using the general positioning of ligands to SH2 (1Kuriyan J. Cowburn D. Curr. Opin. Struct. Biol. 1993; 3: 828-837Crossref Scopus (107) Google Scholar) as a framework, the likely orientation of the 2BP-1 ligand, PVY*ENVamide(10Zhu G. Decker S.J. Maclean D. McNamara D.J. Singh J. Sawyer T.K. Saltiel A.R. Oncogene. 1994; 9: 1379-1385PubMed Google Scholar), was modeled. In an SH(32) model (Fig. 1), these ligands are closest at their C termini, and there is more than 20 Å separating them; about 32 Å separate the C terminus of the SH3 ligand from the N terminus of the SH2 ligand. An initial design of a consolidated ligand for both domains uses this model. Consolidated ligand I (Table 1) then joins the C termini of the individual 2BP and 3BP ligands using a branch through the side chain of a lysine residue. By model building, the methylene segments of the lysyl side chain and the oligoglycyl linker are expected to provide sufficient separation between the C termini of the individual ligands. A series of consolidated ligands, with different ligand segments for SH3, different lengths and orientations of the linker, and different analogs of the phosphotyrosyl residue in the ligand for SH2, were synthesized and tested for affinity to Abl SH(32), and in some cases to SH3 and SH2.TABLE I Open table in a new tab This design and synthesis of consolidated ligands is shown to be effective in Table 1. Increases in affinity of approximately 2 orders of magnitude were observed compared to unbranched equivalents (IversusII, IVversusV). An order of magnitude increase was observed comparing the most strongly bound single ligand to the equivalent consolidated ligand (2BP1versusIV). The affinity of a consolidated ligand changed when a subligand was modified proportionally to the change of affinity of separate ligand (I/3BP-1versusIV/3BP-2). The simple linker chemistry used did not interfere with the subligands affinities to the individual SH3 or SH2 domains. Experiments with the single ligands as inhibitors show classical competitive binding. The values of affinities of ligands with variable linker lengths (III-G5, IV-G6, VI-G7, VII-G8) are most readily interpreted as indicating an optimal length of about G6, with little interaction between the linker and the SH(32) protein. Analogs of the 2BP ligand of IV, namely the H-phosphonate (VIII), and the pY®Y of IV (IX) lead to decreases in affinity. The simplestmodel of how the consolidated ligand binds is that there are specific contacts at the subligand sites, and a solvated linker region. Using a model derived from NMR studies of the SH3 and SH2 domains(6Gosser Y.Q. Zheng J. Overduin M. Mayer B.J. Cowburn D. Structure. 1995; 3: 1075-1088Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), assuming that the orientation for Abl is similar to that seen in Lck(4Eck M.J. Atwell S.K. Shoelson S.E. Harrison S.C. Nature. 1994; 368: 764-769Crossref PubMed Scopus (240) Google Scholar), the two subligands are correctly accommodated, and no specific interactions are forced on the linker segment (Fig. 1). The specificity of interaction is much increased also (Table 1), so that IV will now discriminate between the complete SH(32) and its subdomains by more than an order of magnitude. The decreased affinity of IV compared to 2BP1 for the single SH2 domain is not readily explained. One possibility is that IV forms a partially folded structure reducing the population of ligand in a conformation suitable for binding to SH2. The affinities in Table 1 might be expected, from simple physical chemical concepts, to be somewhat higher for the consolidated ligands. The thermodynamic treatment of the consolidation of fragment affinities has been addressed by Jencks(24Jencks W.P. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 4046-4050Crossref PubMed Scopus (848) Google Scholar). Briefly, for affinities to SH2, SH3, and SH32, the free energy contributions may be related by ΔG°32 = ΔGi2+ΔGi3+ΔGS where the subscripts 32, 2, and 3 refer to the SH domain interaction, and the superscript i refers to the individual binding contributions, and ΔGS represents the change in probability of binding that results from the connection of the two ligands. It may be assumed that the principal method by which apparent affinity increases for the ligands of this work is the reduction in degrees of translational freedom, when one subligand is bound(24Jencks W.P. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 4046-4050Crossref PubMed Scopus (848) Google Scholar). For perfect linker length and geometry, a consolidated ligand affinity up to the product of the individual association constants might be expected (i.e. ΔG°23 = ΔGi2+ΔGi3). Increases in degrees of rotational freedom associated with each glycyl residue, and with the lysyl methylenes reduce this affinity considerably, that is ΔGS represents a large, energetically unfavorable, increase in entropic contribution, and the overall free energy of interaction of the consolidated ligand is enhanced, but not dramatically, over that of its components. There are many possible applications of these consolidated ligands and their analogs. The examples in Table 1 show that for Abl SH(32), the two ligand sites can be oriented as shown in Fig. 1, that the 3BP-1 and 3BP-2 ligands for SH3 (in I and IV) are bound in the same direction, and that the SH2 and SH3 binding sites do not interfere with each other. Applications are expected to other SH(32)-containing proteins for the purpose of similar mapping, or of obtaining high affinity and specificity of binding for investigational or therapeutic purposes(14Brugge J.S. Science. 1993; 260: 918-919Crossref PubMed Scopus (95) Google Scholar). Some examples are dual and higher subligand consolidations for the adaptor protein Grb-2, and reagents like those shown here for Abl, although possibly linear rather than branched(15Feng S. Chen J.K. Yu H. Simon J.A. Schreiber S.L. Science. 1994; 266: 1241-1247Crossref PubMed Scopus (747) Google Scholar), for Src family tyrosyl kinases(16Gilmer T. et al.J. Biol. Chem. 1994; 269: 31711-31719Abstract Full Text PDF PubMed Google Scholar). A major challenge in the investigation of intracellular signal transduction involves the relatively transient nature of the signal transducing complexes formed. Highly specific reagents may permit trapping of, or selective interference with, these complexes. The subligands in such consolidated ligands may be extended to other components of signal transducing complexes, for example the active sites of kinases, or binding sites of pleckstrin homology domains. For Abl, the consolidated ligands shown here are possible early leads to more complex ligands, which may have sufficient affinity and specificity to block the Bcr-Abl fusion kinase that is the predominant pathogenic agent in chronic myologenous leukemia. The chemistry used in these consolidated ligands is relatively simple and was selected to provide flexible linkers, as has been done for the hirulogs(17Maraganore J.M. Bourdon P. Jablonski J. Ramachandran K.L. Fenton II, J.W. Biochemistry. 1990; 29: 7095-7101Crossref PubMed Scopus (443) Google Scholar). Linkers of the correct size and rigidity should provide greater affinities. The structure of the Abl SH(32)/consolidated ligand complex may permit rational design of linkers of an optimal rigidity and size, and also permit identification of additional interactions for increased affinity. Peptidomimetic and non-peptidic linkers are obviously practical, as are other subligands including lipids, steroids, carbohydrates, and nucleic acids. These consolidated ligands present novel opportunities for chemistry at the interface with biology. D. C. thanks Y. Gosser, D. Fushman, S. Cahill, J. Manning, J. Kuriyan, S. K. Burley, and B. Chait for discussion.
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