Discovery of a novel series of potent and selective substrate-based inhibitors of p60c-src protein tyrosine kinase: conformational and topographical constraints in peptide design.

On the basis of the efficient substrate for p60c-src protein tyrosine kinase (PTK) YIYGSFK-NH2 (1) (Km = 55 microM) obtained by combinatorial methods, we have designed and synthesized a series of conformationally and topographically constrained substrate-based peptide inhibitors of this enzyme, which showed IC50 values in the low-micromolar range (1-3 microM). A "rotamer scan" was performed by introducing the four stereoisomers of beta-Me(2')Nal in the postulated interaction site of the peptide inhibitor 23(IC50 = 1.6 microM). This substitution led to selective and potent inhibitors of p60c-src PTK; however, no substantial difference in potency was observed among them. This and the results of the "stereochemical scan" performed at residues 2 and 7 of 3 (peptides 19-21), which form the disulfide bond, may suggest that the enzyme active site does not have rigid topographic requirements and thus is able to achieve important conformational changes to bind the ligand as long as the pharmacophore pattern in the inhibitor is conserved. Two new potent iodo-containing nonphosphorylatable tyrosine analogues were also incorporated into our lead inhibitory sequence 23, producing the most potent inhibitors for p60c-src PTK identified thus far in our studies. Compounds 29 and 30 exhibit IC50 values of 0.13 and 0.54 microM, respectively. Peptide 29 is 420-fold more potent than the parent peptide 1. Selectivity studies of peptides 23-30 toward p60c-src, Lyn, and Lck PTK showed in general high Lyn/Src and moderate Lck/Src selectivity ratios. We found that the chi1 space constraints of the specialized amino acids, introduced at position 3 of the peptide lead 23, were not as important as the configuration of the Calpha of that residue to recognize the subtle chemical environment surrounding the active site of Src and Lck PTK, as reflected on the obtained Lck/Src selectivity ratios.

Interactions of the subunits of smooth muscle myosin phosphatase.

Myosin phosphatase from smooth muscle consists of a catalytic subunit (PP1c) and two non-catalytic subunits, M130 and M20. Interactions among PP1c, M20, and various mutants of M130 were investigated. Using the yeast two-hybrid system, PP1c was shown to bind to the NH2-terminal sequence of M130, 1-511. Other interactions were detected, i.e. PP1c to PP1c, M20 to the COOH-terminal fragment of M130, and dimerization of the COOH-terminal fragment of M130. Mutants of M130 were constructed to localize the PP1c and light chain binding regions. Results from the two-hybrid system indicated two binding sites for PP1c on M130: one site in the NH2-terminal 38 residues and a weaker site(s) in the ankyrin repeats region. Inhibition of PP1c activity with phosphorylase a by the M130 mutants also was consistent with the assignment of these two sites. Overlay assays showed binding of phosphorylated light chain to the ankyrin repeats, probably in the COOH-terminal repeats. Activation of PP1c with phosphorylated light chain required binding sites for PP1c and substrate, plus an additional sequence COOH-terminal to the ankyrin repeats. Thus, activation of phosphatase and binding of PP1c and substrate are properties of the NH2-terminal one-third of M130.

Effect of complexes of ADP and phosphate analogs on the conformation of the Cys707-Cys697 region of myosin subfragment 1.

Recent crystallographic studies have suggested structural differences between the complexes of S1.Mg.ADP with the phosphate analogs aluminium fluoride (AlF4-), vanadate (VO(4)3-) and beryllium fluoride (BeFx) [Fisher, A. J., Smith, C. A., Thoden, J. B., Smith, R., Sutoh, K., Holden, H. M. & Rayment, I. (1995) Biochemistry 34, 8960-8972; Smith, R. & Rayment, I. (1996) Biochemistry 35, 5404-5417]. In this work, chemical modifications, namely labeling of Cys707 (the reactive SH1 thiol) and Cys707-Cys697 (SH1-SH2) cross-linking, were used to compare the S1.ADP.BeFx, S1.ADP. AlF4- and S1.ADP-VO(4)3- complexes with specific states of the myosin-ATPase pathway. Modification of Cys707 with the fluorescent monofunctional reagents 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin and N-iodoacetyl-N'-(5-sulfo-1-naphtyl)ethylenediamine has shown that the reactivity of the SH1 group depends on the nucleotide bound to S1. The observed rates of Cys707 modification at 20 degrees C lead to the conclusion that S1.ADP.BeFx is similar to S1*.ATP, while S1.ADP.AlF4- and S1.ADP.VO(4)3- are more similar to S1**.ADP.Pi. The conformations of the analog states were also compared by monitoring the dissociation of the fluorescent nucleotide analog 1-N6-ethenoadenosine diphosphate (ADP[C2H2]) from the active site of Cys707-modified (by N-ethylmaleimide) and Cys707-Cys697-cross-linked (by N,N'-p-phenylene dimaleimide) S1.ADP[C2H2].AlF4- and S1.ADP[C2H2]. BeFx. Our results suggest that the conformations of the S1.ADP.AlF4-, S1.ADP.VO(4)3- and S1.ADP.BeFx complexes in the Cys707-Cys697 region are distinct from each other, with the former two at least partially resembling the S1**.ADP.Pi state, while the latter is similar to the prehydrolyzed S1*.ATP state.

Complexes of myosin subfragment-1 with adenosine diphosphate and phosphate analogs: probes of active site and protein conformation.

Previous work has revealed phosphate-dependent differences in the complexes formed from myosin subfragment-1 with adenosine diphosphate (S1.ADP) and aluminum fluoride (AlF4-) or beryllium fluoride (BeFx) [Phan and Reisler, Biophys. J., 66 (1994) A78], with the former resembling more the S1**.ADP.Pi state while the latter resembles more the S1.ATP state. In this work, the conformations of the S1.epsilon ADP.AlF4- and S1.epsilon ADP.BeFx, complexes were examined by nucleotide chase and collisional quenching experiments. epsilon ADP release from S1.epsilon ADP.AlF4- was slower than that from S1.epsilon ADP.BeFx. However, acrylamide titrations of S1.epsilon ADP.AlF4- and S1.epsilon ADP.BeFx showed little difference in nucleotide protection from quenching between the two complexes. This contrasts with the earlier observation on phosphate analog-dependent changes in the reactivity of the SH1 group on S1. To confirm phosphate-related perturbation of the SH1-SH2 sequence, emission spectra of fluorescein (IAF)-labeled SH1 and IANBD-labeled SH2 were recorded for S1 complexes with nucleotides and phosphate analogs. Considerable differences were found between the BeFx and AlF4- complexes with S1.MgADP for both SH1- and SH2-labeled proteins. These results are consistent with a recent crystallographic study of S1 complexes with ADP and phosphate analogs [Fisher et al., Biophys. J., 68 (1995) 19S] and the idea that the opening of the nucleotide cleft on S1 does not change much during ATP hydrolysis [Franks-Skiba et al., Biochemistry, 33 (1994) 12720], while significant changes in the SH1-SH2 region accompany phosphate cleavage.

Extensively methylated myosin subfragment-1: examination of local structure, interactions with nucleotides and actin, and ligand-induced conformational changes.

The atomic structure of myosin subfragment-1 (S1) has been recently solved for crystals of extensively methylated S1 [Rayment et al. (1993) Science 261, 50-58]. In this study, the effect of such a modification on S1 structure and function was examined. According to the far- and near-ultraviolet CD spectra, the methylation does not affect the secondary structure of S1 but causes limited changes in its tertiary structure. The methylation significantly decreases the affinity of S1 for actin in rigor and, to a lesser degree, that of S1 to actin in the presence of MgATP gamma S. This modification, like the trinitrophenylation of Lys-83, accelerates the dissociation of a nucleotide trapped on S1 either by phosphate analogs or by cross-linking of the SH1 and SH2 thiols. Methylation strongly impairs the coupling between the actin- and nucleotide-binding sites as revealed by the reduced effect of actin on the release of epsilon ADP from the active site. It also causes a complete loss of in vitro motility of actin filaments over methylated HMM. In addition to this, methylation also impairs the communication between other sites on S1 including that between the nucleotide-binding site and SH1, and the actin-binding site and the 27/50 kDa junction and a site at 74 kDa from the N-terminus of S1. These changes are revealed in SH1 modification, thermolysin digestion, and vanadate-dependent photocleavage experiments, respectively. The increased rate of thermal denaturation of S1 and the loss of S1 protection by ADP and actin from this process also indicate flawed communications in methylated S1. It is concluded that these relatively mild but numerous and important changes impair the function of methylated S1.

Dynamic properties of actin. Structural changes induced by beryllium fluoride.

Beryllium fluoride (BeFx) has been widely used as a phosphate analogue in nucleotide-binding proteins. It was found to bind tightly to F- but not G-actin (Combeau C., and Carlier M. F. (1988) J. Biol. Chem. 263, 17429-17436) and to affect the three-dimensional structure of filaments by stabilizing the subdomain 2 region of the actin promoter (Orlova, A., and Egelman, E. H. (1992) J. Mol. Biol. 227, 1043-1053). In this work we examined the BeFx-induced structural and functional changes in G- and F-actin by using proteolysis, chemical modifications, ATPase, and in vitro motility assays. The results of proteolysis studies show that BeFx binds also to MgADP-G-actin and renders its subdomain 2 region more similar to that in MgATP-G-actin. This is manifested in enhanced subtilisin and decreased tryptic digestions in subdomain 2 of G-actin. BeFx had a strong effect on the proteolysis of MgADP-F-actin: both the tryptic and subtilisin digestions in subdomain 2 were completely inhibited. Significant protection against proteolysis in this region was observed even at 1:14 molar ratios of BeFx to actin indicating cooperative effects on the structure of the actin filament. A similar although milder effect of phosphate on the proteolysis of F-actin suggests that BeFx acts as a phosphate analogue in this system. BeFx also induces changes in the subdomain 1 region of F-actin. This is revealed via reduced rates of Cys-374 alkylation with 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin and an increased subtilisin cleavage near the C terminus of actin in the presence of BeFx. The BeFx-induced structural changes in actin have little effect on its interactions with myosin. BeFx inhibits only slightly the actin-activated ATPase activity of S1 by decreasing Vmax without affecting KM. Additionally, the binding of BeFx to actin does not change the sliding velocity of actin filaments in the in vitro motility assays. The BeFx-induced specific and distinct changes in G- and F-actin point to the dynamic nature of actin structure and the local differences between monomeric and polymeric forms of actin.

Aluminum fluoride interactions with troponin C.

The increasing interest in the metal ion aluminum fluoride and beryllium fluoride complexes as phosphate analogs in the myosin ATPase reaction and in muscle fiber studies prompted the examination of their interactions with the regulatory system of troponin and tropomyosin. In this work, the effects of these metal ion analogs on the spectral properties of the Ca(2+)-binding subunit of troponin, troponin C (TnC), were examined. In contrast to beryllium fluoride which did not change the spectral properties of TnC, aluminum fluoride binding induced an increase in both the alpha-helicity and the tyrosine fluorescence of TnC and exposed a hydrophobic region on this protein for fluorescent probe binding. Aluminum fluoride also reduced the Ca2+ and/or Mg(2+)-induced changes on TnC. These results indicate a direct interaction of aluminum fluoride with TnC and merit consideration in designing muscle fiber experiments with this phosphate analog.

Kinetic and equilibrium analysis of the interactions of actomyosin subfragment-1.ADP with beryllium fluoride.

The hypothesis that the stable ternary complex formed between myosin subfragment-1, MgADP and beryllium fluoride (BeF3-), denoted S-1 not equal to .ADP.BeF3-, is an analog of the intermediate state S-1**.ADP.P(i) has been tested in this work by examining the interactions of S-1 not equal to .ADP.BeF3- with actin. Equilibrium binding measurements revealed that actin bound weakly to the S-1 not equal to .ADP.BeF3- complex (Ka = 10(4) M-1) in the presence of 40 mM KCl. The stability of this complex was strongly salt-dependent. The association constant of BeF3- to the acto-S-1.ADP complex (KBe approximately 10(3) M-1) was 100-fold weaker than its binding to the S-1.ADP complex. While inhibiting the S-1 ATPase strongly, BeF3- had no effect on the Vmax value (10 +/- 1.0 s-1) of the actin-activated ATPase of S-1. The rates of BeF3- binding and dissociation from the acto-S-1.ADP.BeF3- complex were determined by stopped-flow measurements. The hyperbolic dependence of the rates of BeF3- binding to acto-S-1.ADP (kobs) on BeF3- concentrations suggested that the acto-S-1.ADP.BeF3- complex was formed in at least two steps: binding followed by isomerization. The binding constant was 1.2 x 10(3) M-1, and the maximum kobs was 2.5 s-1. The dissociation of BeF3- from the acto-S-1.ADP.BeF3- complex was monitored via decrease in the fluorescence of 1-N6-ethenoadenosine diphosphate (epsilon ADP). The fluorescence decrease fitted two exponential terms.(ABSTRACT TRUNCATED AT 250 WORDS)