We developed an Escherichia coli cell-based system to generate proteins containing 3-iodo-L-tyrosine at desired sites, and we used this system for structure determination by single-wavelength anomalous dispersion (SAD) phasing with the strong iodine signal. Tyrosyl-tRNA synthetase from Methanocaldococcus jannaschii was engineered to specifically recognize 3-iodo-L-tyrosine. The 1.7 A crystal structure of the engineered variant, iodoTyrRS-mj, bound with 3-iodo-L-tyrosine revealed the structural basis underlying the strict specificity for this nonnatural substrate; the iodine moiety makes van der Waals contacts with 5 residues at the binding pocket. E. coli cells expressing iodoTyrRS-mj and the suppressor tRNA were used to incorporate 3-iodo-L-tyrosine site specifically into the ribosomal protein N-acetyltransferase from Thermus thermophilus. The crystal structure of this enzyme with iodotyrosine was determined at 1.8 and 2.2 Angstroms resolutions by SAD phasing at CuK alpha and CrK alpha wavelengths, respectively. The native structure, determined by molecular replacement, revealed no significant structural distortion caused by iodotyrosine incorporation.

The Rac-specific guanine nucleotide exchange factor (GEF) Asef is activated by binding to the tumor suppressor adenomatous polyposis coli mutant, which is found in sporadic and familial colorectal tumors. This activated Asef is involved in the migration of colorectal tumor cells. The GEFs for Rho family GTPases contain the Dbl homology (DH) domain and the pleckstrin homology (PH) domain. When Asef is in the resting state, the GEF activity of the DH-PH module is intramolecularly inhibited by an unidentified mechanism. Asef has a Src homology 3 (SH3) domain in addition to the DH-PH module. In the present study, the three-dimensional structure of Asef was solved in its autoinhibited state. The crystal structure revealed that the SH3 domain binds intramolecularly to the DH domain, thus blocking the Rac-binding site. Furthermore, the RT-loop and the C-terminal region of the SH3 domain interact with the DH domain in a manner completely different from those for the canonical binding to a polyproline-peptide motif. These results demonstrate that the blocking of the Rac-binding site by the SH3 domain is essential for Asef autoinhibition. This may be a common mechanism in other proteins that possess an SH3 domain adjacent to a DH-PH module.The Rho family GTPases, including Rho, Rac, and Cdc42, participate in actin cytoskeletal network reorganization, thus resulting in cell migration and cell-cell adhesion (1). The Rho family GTPases are activated by guanine nucleotide exchange factors (GEFs), 3 which possess the Dbl homology (DH) domain responsible for the GEF activity. The DH domain is followed by the PH domain, and a number of DH-PH fragment structures have been reported (2). The Asef protein contains the DH and PH domains (Fig. 1a) and exhibits a Rac-specific GEF activity when it is bound to the armadillo repeat of the tumor suppressor adenomatous polyposis coli (APC) with a mutation (Asef stands for APC-stimulated guanine nucleotide exchange factor) (3). The Asef protein bound with the mutated APC is involved in the migration of colorectal tumor cells (4). The N-terminal region of Asef includes the APC-binding region (ABR) (Fig. 1a), which is presumably a flexible peptide segment. In contrast, Asef by itself showed very weak GEF activity (3). On the other hand, a mutant form of Asef, lacking the N-terminal region, displayed GEF activity as strong as that of the APCstimulated, full-length Asef. Therefore, the N-terminal region "autoinhibits" the DH domain of Asef in isolation, but the molecular mechanism of the autoinhibition has been elusive. A Src-homology 3 (SH3) domain resides between the ABR and the DH domain (Fig. 1a). EXPERIMENTAL PROCEDURESProtein Expression and Purification-Selenomethionine-labeled Asef (residues 66 -540) with an N-terminal histidine tag was expressed in the cell-free expression system (5). The protein was purified by chromatography on a HisTrap column (GE Healthcare) and was subjected to tobacco etch virus protease digestion. The Asef protein was subsequently purified by MonoQ and Superdex75 ...

The DNA polymerase processivity factor of the Epstein-Barr virus, BMRF1, associates with the polymerase catalytic subunit, BALF5, to enhance the polymerase processivity and exonuclease activities of the holoenzyme. In this study, the crystal structure of C-terminally truncated BMRF1 (BMRF1-⌬C) was solved in an oligomeric state. The molecular structure of BMRF1-⌬C shares structural similarity with other processivity factors, such as herpes simplex virus UL42, cytomegalovirus UL44, and human proliferating cell nuclear antigen. However, the oligomerization architectures of these proteins range from a monomer to a trimer. PAGE and mutational analyses indicated that BMRF1-⌬C, like UL44, forms a C-shaped head-to-head dimer. DNA binding assays suggested that basic amino acid residues on the concave surface of the C-shaped dimer play an important role in interactions with DNA. The C95E mutant, which disrupts dimer formation, lacked DNA binding activity, indicating that dimer formation is required for DNA binding. These characteristics are similar to those of another dimeric viral processivity factor, UL44. Although the R87E and H141F mutants of BMRF1-⌬C exhibited dramatically reduced polymerase processivity, they were still able to bind DNA and to dimerize. These amino acid residues are located near the dimer interface, suggesting that BMRF1-⌬C associates with the catalytic subunit BALF5 around the dimer interface. Consequently, the monomeric form of BMRF1-⌬C probably binds to BALF5, because the steric consequences would prevent the maintenance of the dimeric form. A distinctive feature of BMRF1-⌬C is that the dimeric and monomeric forms might be utilized for the DNA binding and replication processes, respectively.The Epstein-Barr virus (EBV), 4 a human herpesvirus harboring a 172-kbp dsDNA genome, is associated with several B-cell and epithelial cell malignancies and can choose between two alternative life cycles, latent and lytic infection (1). The EBV genomes are replicated as circular plasmid molecules, using the cellular replication machinery of the host in the latent phase of the viral life cycle. On the other hand, after the induction of lytic viral replication, the EBV genome is amplified 100 -1,000-fold by the viral replication machinery. The replication intermediates are large head-to-tail concatemers resulting from rolling-circle DNA replication initiated from oriLyt (2). The EBV replication machinery consists of seven viral gene products (3) as follows: the BZLF1 protein, an oriLytbinding protein; the BALF5 protein, a DNA polymerase catalytic subunit; the BMRF1 protein, a polymerase processivity factor; the BALF2 protein, a single-stranded DNA-binding protein; and the BBLF4, BSLF1, and BBLF2/3 proteins, putative helicase, primase, and helicase-primase-associated proteins, respectively. It has been suggested that all of the proteins, except for the BZLF1 protein, work together at replication forks to synthesize the leading and lagging strands of the concatemeric EBV genome (2). The EBV DNA polymerase holoenzy...

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Leucyl/phenylalanyl-tRNA-protein transferase (L/F-transferase) is an N-end rule pathway enzyme, which catalyzes the transfer of Leu and Phe from aminoacyl-tRNAs to exposed N-terminal Arg or Lys residues of acceptor proteins. Here, we report the 1.6 Å resolution crystal structure of L/F-transferase (JW0868) from Escherichia coli, the first three-dimensional structure of an L/F-transferase. The L/Ftransferase adopts a monomeric structure consisting of two domains that form a bilobate molecule. The N-terminal domain forms a small lobe with a novel fold. The large C-terminal domain has a highly conserved fold, which is observed in the GCN5-related N-acetyltransferase (GNAT) family. Most of the conserved residues of L/F-transferase reside in the central cavity, which exists at the interface between the N-terminal and C-terminal domains. A comparison of the structures of L/F-transferase and the bacterial peptidoglycan synthase FemX, indicated a structural homology in the C-terminal domain, and a similar domain interface region. Although the peptidyltransferase function is shared between the two proteins, the enzymatic mechanism would differ. The conserved residues in the central cavity of L/F-transferase suggest that this region is important for the enzyme catalysis.