Shuichi Kojima

We reported previously that our designed polypeptide 3 (21 residues), which has three repeats of a seven-amino-acid sequence (LETLAKA)(3), forms not only an amphipathic -helix structure but also long fibrous assemblies in aqueous solution. To address the relationship between the electrical states of the polypeptide and its -helix and fibrous assembly formation, we characterized mutated polypeptides in which charged amino acid residues of 3 were replaced with Ser. We prepared the following polypeptides: 2S3 (LSTLAKA)(3), in which all Glu residues were replaced with Ser residues; 6S3 (LETLASA)(3), in which all Lys residues were replaced with Ser; and 2S6S3 (LSTLASA)(3); in which all Glu and Lys residues were replaced with Ser. In 0.1M KCl, 2S3 formed an -helix under basic conditions and 6S3 formed an -helix under acid conditions. In 1M KCl, they both formed -helices under a wide pH range. In addition, 2S3 and 6S3 formed fibrous assemblies under the same buffer conditions in which they formed -helices. -Helix and fibrous assembly formation by these polypeptides was reversible in a pH-dependent manner. In contrast, 2S6S3 formed an -helix under basic conditions in 1M KCl. Taken together, these findings reveal that the charge states of the charged amino acid residues and the charge state of the Leu residue located at the terminus play an important role in -helix formation.

We previously characterized alpha 3, a polypeptide that has a three times repeated sequence of seven amino acids (abc-defg: LETLAKA) and forms fibrous assemblies composed of amphipathic alpha-helices. Upon comparison of the amino acid sequences of alpha 3 with other alpha-helix forming polypeptides, we proposed that the fibrous assemblies were formed due to the alanine (Ala) residues at positions e and g. Here, we characterized seven alpha 3 analog polypeptides with serine (Ser), glycine (Gly), or charged residues substituted for Ala at positions e and g. The alpha-helix forming abilities of the substituted polypeptides were less than that of alpha 3. The polypeptides with amino acid substitutions at position g and the polypeptide KE alpha 3, in which Ala was substituted with charged amino acids, formed few fibrous assemblies. In contrast, polypeptides with Ala replaced by Ser at position e formed beta-sheets under several conditions. These results show that Ala residues at position e and particularly at position g are involved in the formation of fibrous assemblies. (C) 2014 Wiley Periodicals, Inc.

L-Amino acid ligase (Lal) catalyzes the formation of a dipeptide from two L-amino acids in an ATP-dependent manner and belongs to the ATP-grasp superfamily. Bacillus subtilis YwfE, the first identified Lal, produces the dipeptide antibiotic bacilysin, which consists of L-Ala and L-anticapsin. Its substrate specificity is restricted to smaller amino acids such as L-Ala for the N-terminal end of the dipeptide, whereas a wide range of hydrophobic amino acids including L-Phe and L-Met are recognized for the C-terminal end in vitro. We determined the crystal structures of YwfE with bound ADP-Mg2+-Pi and ADP-Mg2+-L-Ala at 1.9 and 2.0 angstrom resolutions, respectively. On the basis of these structures, we generated point mutants of residues that are considered to participate in the recognition of L-Ala and measured their ATPase activity. The conserved Arg328 is suggested to be a crucial residue for L-Ala recognition and catalysis. The mutation of Trp332 to Ala caused the enzyme to hydrolyze ATP, even in the absence of L-Ala, and the structure of this mutant protein appeared to show a cavity in the N-terminal substrate-binding pocket. These results suggest that Trp332 plays a key role in restricting the substrate specificity to smaller amino acids such as L-Ala. Moreover, Trp332 mutants can alter the substrate specificity and activity depending on the size and shape of substituted amino acids. These observations provide sufficient scope for the rational design of Lal to produce desirable dipeptides. We propose that the positioning of the conserved Arg residue in Lal is important for enantioselective recognition of L-amino acids.

Polypeptide alpha 3 (21 residues), with three repeats of a seven-amino-acid sequence (LETLAKA)(3), forms an amphipathic alpha-helix and a long fibrous assembly. Here, we investigated the ability of alpha 3-series polypeptides (with 14-42 residues) of various chain lengths to form alpha-helices and fibrous assemblies. Polypeptide alpha 2 (14 residues), with two same-sequence repeats, did not form an alpha-helix, but polypeptide alpha 2L (15 residues; alpha 2 with one additional leucine residue on its carboxyl terminal) did form an alpha-helix and fibrous assembly. Fibrous assembly formation was associated with polypeptides at least as long as polypeptide alpha 2L and with five leucine residues, indicating that the C-terminal leucine has a critical element for stabilization of alpha-helix and fibril formation. In contrast, polypeptides alpha 5 (35 residues) and alpha 6 (42 residues) aggregated easily, although they formed alpha-helices. A 15-35-residue chain was required for fibrous assembly formation. Electron microscopy and X-ray fiber diffraction showed that the thinnest fibrous assemblies of polypeptides were about 20 angstrom and had periodicities coincident with the length of the alpha-helix in a longitudinal direction. These results indicated that the alpha-helix structures were orientated along the fibrous axis and assembled into a bundle. Furthermore, the width and length of fibrous assemblies changed with changes in the pH value, resulting in variations in the charged states of the residues. Our results suggest that the formation of fibrous assemblies of amphipathic alpha-helices is due to the assembly of bundles via the hydrophobic faces of the helices and extension with hydrophobic noncovalent bonds containing a leucine.

Bacillus subtilis YwfE, an l-amino-acid ligase, catalyzes the formation of an a-dipeptide from l-amino acids in an ATP-dependent manner. In order to elucidate the substrate-recognition mode and the reaction mechanism of this ligase, native and selenomethionine-derivatized (SeMet) crystals of YwfE in the presence of ADP, MgCl2 and the dipeptide l-Ala-l-Gln were obtained using the similar to hanging-drop vapour-diffusion method. These crystals diffracted to 1.9 and 2.8 angstrom resolution, respectively. Preliminary SAD phase calculations using the data set from the SeMet crystal suggested that the crystal belonged to the hexagonal space group P6522, with unit-cell parameters a = b = 90.85, c similar to=similar to 250.31 angstrom, and contained one molecule in the asymmetric unit with a solvent content of 57.3%.