Conformation of thymosin β₄ in water determined by NMR spectroscopy

Abstract

The conformational preferences of a 43-amino-acid G-actin-binding peptide, thymosin P4, in water at 1, 4 and 14°C and at pH 3.0 and 6.5 were studied by NMR. NMR showed that thymosin p4 lacks a uniquely folded conformation in water. However, some preferential a-helical conformations of thymosin /I4 can be observed in aqueous solutions. The segment at residues 5-16 showed characteristic interactions for conformations in both the P-strand and a-helical regions of the 4-y space, based on strong CH(i)-NH(i+ 1) interactions and NH-NH, C"H(i)-NH(i+3), and C"H(i)-CpH(i+3) interactions, respectively. At 1 -4"C, another segment at residues 31 -37 also shows both p and a conformations, forming however a less well-defined helix than the segment at residues 5 -16. At 14"C, the conformational population of the helix at positions 5-16 is shifted more towards the random and turn-like structures, whereas the segment at positions 31 -37 becomes exclusively a random coil.The cytoplasmic salt conditions in a non-muscle cell strongly favor the polymerization of actin and only recently has it been determined how cells manage to keep more than 50% of the actin pool in an unpolymerized state. It is now thought that thymosin P4 is the main actin-sequestering peptide, which forms a 1 : 1 complex with actin and thus shifts the polymerization equilibrium from filamentous (F-actin) to globular actin (G-actin;Weeds and Way, 1991;Safer, 1992). Furthermore, the interaction of thymosin and actin inhibits of ADP/ATP exchange in actin, consequently leading to a high concentration of ADP-actin, which has a drastically decreased tendency to form actin filaments. Profilins, another class of G-actin-binding proteins, increase the nucleotide exchange and are thought to compete with thymosin in this reaction (Goldschmidt-Clermont et al., 1992).Since the elucidation of the crystal structure of actin (Kabsch et al., 1990), it is of prime importance to determine the structures of actin-binding proteins and to characterize the nature of the protein/protein interactions. Recently, we determined the NMR structure of hisactophilin, a histidinerich actin-binding protein (Scheel et al., 1989;Habazettl et al., 1992), and of thymosin p4 (Zarbock et al., 1990). The latter, however, was obtained in solutions containing fluorinated alcohols, i.e. conditions which might not reflect the true conformation of the protein in the aqueous environment. We present in this study the determination of the solution conformation of thymosin / I 4 in water by 'H-NMR spectroscopy. Using a variety of two-dimensional (2D) NMR techniques (Emst et al., 1987), the 'H-NMR spectra were assigned in a sequential manner (Wuthrich, 1986 spectra formed the basis for the determination of the conformational preferences of thymosin p4 at different temperatures and pH. MATERIALS AND METHODS Sample preparation of thymosin p4Thymosin P4 was isolated as described by Zarbock et al. (1990). The NMR samples of thymosin P4 were dissolved in 90% H,0/10% D,O at an approximate concentration of 2 mM peptide...

Ablation of cyclase-associated protein 2 (CAP2) leads to cardiomyopathy

“…These proteins can either sequester monomeric actin or inhibit actin polymerization like thymosin or nucleate actin assembly like Spire, Cordon-bleu, and JMY [10]. WH2 motifs are intrinsically disordered, adopting an a-helical structure only upon binding to actin [11]. CAP in lower eukaryotes as well as mouse CAP1 are involved in cell polarity, motility, and receptor-mediated endocytosis [12][13][14].…”

A Phage Display-based Method for Determination of Relative Affinities of Mutants

“…WT, WT-like activity (ϩ), mutants that are not displayed (ND), and displayed mutants with no observed binding (Ϫ) are indicated. Second, NMR experiments showed that the motif in noncomplexed thymosin ␤4 is structurally poorly defined (17,18), whereas algorithms predict a ␣-helical conformation We show that at certain positions in the motif, some substitutions yield better or wild type binders. If we correlate this with the secondary structure propensities of these mutant amino acids (43), we can speculate on the local conformation of the hexapeptide motif in the actin bound configuration.…”

“…Thymosin ␤4 interacts with actin via residues in an ␣-helix and a conserved hexapeptide motif (LKKTET) (14 -16). NMR studies of thymosin ␤4 reveal no unique structure for this motif; however, it is evident that the motif must become structured upon binding actin (17)(18)(19). A seemingly related sequence (LKKEKG) is present in a set of C-terminal headpiece domains (15,16) implicated in F-actin binding (20).…”

Identification of Contact Sites in the Actin-Thymosin β4 Complex by Distance-dependent Thiol Cross-linking

“…In a detailed study, Vancompernolle et al (9) have shown that binding of T␤4 to actin is mainly mediated by the hexamotif LKKTET (17)(18)(19)(20)(21)(22), since loss of this sequence is paralleled by an almost complete loss of inhibitory activity. Alterations in the N-terminal part (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16) of the peptide strongly influence the inhibitory activity of T␤4, whereas alterations in the C-terminal part (31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43) seem to be of minor importance (9). As shown by 1 H NMR spectroscopy (10, 11) T␤4 does not contain an ordered conformation in aqueous solution but tends to form an ␣-helical conformation between residues 5 and 16 (11).…”