Thus, the Laz mutant with a pectate lyase B (cells showed a single protein band of 22?kDa around the SDS-PAGE gel (Fig. structural features of the H.8 moiety in Laz using X-ray crystallography and demonstrate that while the azurin moiety of Laz adopts a -sandwich fold with 2 -sheets arranged in the Greek key motif, the H.8 epitope was present as a disordered structure outside the Greek key motif. Structures of Paz and H.8 epitope-deficient Laz are well superimposed. The structural flexibility of the H.8 motif in Laz explains the extracellular location of Laz in Neisseria where it can bind the key components of brain tumor cells to disrupt their tight junctions and allow entry of Laz inside the tumors to exert cytotoxicity. secrete azurin extracellularly in response to the presence of various human cancer cells (e.g., melanoma and breast cancer cells).3 Azurin exhibits significant cytotoxicity against cancer cells, while little cytotoxicity is observed against normal cells.4 The protein can preferentially enter cancer cells and bind to the tumor suppressor protein p53.5 The complex formed by azurin and p53 as well as its electron transfer partner cytochrome induces apoptosis in cancer cells through cell cycle arrest at the G1 phase or caspase-mediated mitochondrial cytochrome release.6,7 Azurin is therefore expected to be a potential drug for cancer therapy.8,9 The precursor form of azurin (148 residues) produced by is converted to a mature form (Paz, 128 residues) through the release of a signal peptide (20 residues) across the inner membrane (Fig. 1).10 Site-directed mutagenesis and deletion mutations indicate that the presence of a copper ion and redox activity was not essential for the cytotoxicity of Paz.11 The internal sequence of Paz (residue nos. 50C77 in the mature form, termed Deoxycholic acid sodium salt p28) functions as a putative protein transduction domain responsible for Deoxycholic acid sodium salt azurin’s penetration into cancer cells12 through endocytotic and nonendocytotic mechanisms.13 Based on crystal structures, bacterial azurins show structural similarity to ephrinB2, Deoxycholic acid sodium salt a ligand for the receptor tyrosine kinase EphB2.14,15 Cell signaling through Eph/ephrin is involved in cancer progression.14,15 The chemically synthesized C-terminal domain of Paz (residue nos. 96C113 Deoxycholic acid sodium salt in the mature form), which is similar to ephrinB2 at the GH loop region responsible for receptor binding,15 inhibits the growth of various cancer cells.14 Indeed, it has been shown that azurin represses ephrinB2-mediated autophosphorlyation of the EphB2 tyrosine ITSN2 residue, thereby interfering with upstream cell signaling and contributing to cancer cell growth inhibition.14 Open in a separate window Determine 1. Amino acid sequence alignment of Laz and Paz using the CLUSTALW program (http://clustalw.genome.ad.jp/). Laz, azurin; Paz, azurin. Identical and comparable amino acid residues in Laz and Paz are denoted by asterisks and dots, respectively. Letters in italic and strong indicate the signal peptide and H.8 epitope, respectively. The amino acid residues responsible for binding to the metal ion are boxed. The regions corresponding to cylinders and arrows represent -helices and -strands, respectively. The H.8 epitope is intrinsically disordered. Laz used in this study has Ala18 instead of Cys18. In addition to azurin, rusticyanin, another bacterial cupredoxin, causes apoptosis in human cancer cells.16 Neisserial azurin-like protein, a lipobox-dependent lipid-modified azurin (Laz) bound to the outer membrane,17 also enters brain tumors such as glioblastomas and exhibits significant toxicity against the cancer cells,18 suggesting that Laz must cross the entry barrier to reach brain tumor cells. Laz includes a 39-amino acid residue region called the H.8 epitope at the N terminus prior to the azurin domain. In our previous studies with Laz, the H.8 epitope of the protein, but not its lipidation, was demonstrated to be essential for disrupting the entry barrier to provide access to brain tumor cells.18 To obtain clues around the entry of Laz into brain tumor cells, we decided the structure of Laz by X-ray crystallography and our findings are described herein. Results Comparison of primary structure between Laz and Paz Laz consists of 183 amino acid residues with a molecular weight of 18532 (Fig. 1).17 Similar to other bacterial outer membrane-bound lipoproteins, Laz includes a conserved 4-amino acid.
