Genetically encoded caged amino acids can be used to control the dynamics of protein activities and cellular localization in response to external cues. In the present study, we revealed the ...structural basis for the recognition of O-(2-nitrobenzyl)-L-tyrosine (oNBTyr) by its specific variant of Methanocaldococcus jannaschii tyrosyl-tRNA synthetase (oNBTyrRS), and then demonstrated its potential availability for time-resolved X-ray crystallography. The substrate-bound crystal structure of oNBTyrRS at a 2.79 Å resolution indicated that the replacement of tyrosine and leucine at positions 32 and 65 by glycine (Tyr32Gly and Leu65Gly, respectively) and Asp158Ser created sufficient space for entry of the bulky substitute into the amino acid binding pocket, while Glu in place of Leu162 formed a hydrogen bond with the nitro moiety of oNBTyr. We also produced an oNBTyr-containing lysozyme through a cell-free protein synthesis system derived from the Escherichia coli B95. ΔA strain with the UAG codon reassigned to the nonnatural amino acid. Another crystallographic study of the caged protein showed that the site-specifically incorporated oNBTyr was degraded to tyrosine by light irradiation of the crystals. Thus, cell-free protein synthesis of caged proteins with oNBTyr could facilitate time-resolved structural analysis of proteins, including medically important membrane proteins.
Recent advances have fundamentally changed the ways in which synthetic amino acids are incorporated into proteins, enabling their efficient and multiple-site incorporation, in addition to the 20 ...canonical amino acids. This development provides opportunities for fresh approaches toward addressing fundamental problems in bioengineering. In the present study, we showed that the structural stability of proteins can be enhanced by integrating bulky halogenated amino acids at multiple selected sites. Glutathione S-transferase was thus stabilized significantly (by 5.2 and 5.6 kcal/mol) with 3-chloro- and 3-bromo-l-tyrosines, respectively, incorporated at seven selected sites. X-ray crystallographic analyses revealed that the bulky halogen moieties filled internal spaces within the molecules, and formed non-canonical stabilizing interactions with the neighboring residues. This new mechanism for protein stabilization is quite simple and applicable to a wide range of proteins, as demonstrated by the rapid stabilization of the industrially relevant azoreductase.
DOCK2, a hematopoietic cell-specific, atypical guanine nucleotide exchange factor, controls lymphocyte migration through ras-related C3 botulinum toxin substrate (Rac) activation. Dedicator of ...cytokinesis 2–engulfment and cell motility protein 1 (DOCK2{bullet}ELMO1) complex formation is required for DOCK2-mediated Rac signaling. In this study, we identified the N-terminal 177-residue fragment and the C-terminal 196-residue fragment of human DOCK2 and ELMO1, respectively, as the mutual binding regions, and solved the crystal structure of their complex at 2.1-Å resolution. The C-terminal Pro-rich tail of ELMO1 winds around the Src-homology 3 domain of DOCK2, and an intermolecular five-helix bundle is formed. Overall, the entire regions of both DOCK2 and ELMO1 assemble to create a rigid structure, which is required for the DOCK2{bullet}ELMO1 binding, as revealed by mutagenesis. Intriguingly, the DOCK2{bullet}ELMO1 interface hydrophobically buries a residue which, when mutated, reportedly relieves DOCK180 from autoinhibition. We demonstrated that the ELMO-interacting region and the DOCK-homology region 2 guanine nucleotide exchange factor domain of DOCK2 associate with each other for the autoinhibition, and that the assembly with ELMO1 weakens the interaction, relieving DOCK2 from the autoinhibition. The interactions between the N- and C-terminal regions of ELMO1 reportedly cause its autoinhibition, and binding with a DOCK protein relieves the autoinhibition for ras homolog gene family, member G binding and membrane localization. In fact, the DOCK2{bullet}ELMO1 interface also buries the ELMO1 residues required for the autoinhibition within the hydrophobic core of the helix bundle. Therefore, the present complex structure reveals the structural basis by which DOCK2 and ELMO1 mutually relieve their autoinhibition for the activation of Rac1 for lymphocyte chemotaxis.
Transcription of DNA to RNA by DNA-dependent RNA polymerase (RNAP) is the first step of gene expression and a major regulation point. Bacteriophages hijack their host's transcription machinery and ...direct it to serve their needs. The gp39 protein encoded by Thermus thermophilus phage P23-45 binds the host's RNAP and inhibits transcription initiation from its major "-10/-35" class promoters. Phage promoters belonging to the minor "extended -10" class are minimally inhibited. We report the crystal structure of the T. thermophilus RNAP holoenzyme complexed with gp39, which explains the mechanism for RNAP promoter specificity switching. gp39 simultaneously binds to the RNAP β-flap domain and the C-terminal domain of the σ subunit (region 4 of the σ subunit σ4), thus relocating the β-flap tip and σ4. The ~45 Å displacement of σ4 is incompatible with its binding to the -35 promoter consensus element, thus accounting for the inhibition of transcription from -10/-35 class promoters. In contrast, this conformational change is compatible with the recognition of extended -10 class promoters. These results provide the structural bases for the conformational modulation of the host's RNAP promoter specificity to switch gene expression toward supporting phage development for gp39 and, potentially, other phage proteins, such as T4 AsiA.
Receptor activity‐modifying protein (RAMP) 1 forms a heterodimer with calcitonin receptor‐like receptor (CRLR) and regulates its transport to the cell surface. The CRLR·RAMP1 heterodimer functions as ...a specific receptor for calcitonin gene‐related peptide (CGRP). Here, we report the crystal structure of the human RAMP1 extracellular domain. The RAMP1 structure is a three‐helix bundle that is stabilized by three disulfide bonds. The RAMP1 residues important for cell‐surface expression of the CRLR·RAMP1 heterodimer are clustered to form a hydrophobic patch on the molecular surface. The hydrophobic patch is located near the tryptophan residue essential for binding of the CGRP antagonist, BIBN4096BS. These results suggest that the hydrophobic patch participates in the interaction with CRLR and the formation of the ligand‐binding pocket when it forms the CRLR·RAMP1 heterodimer.
Cyclic nucleotide phosphodiesterases (PDEs) catalyze the degradation of the cyclic nucleotides cAMP and cGMP, which are important second messengers. Five of the 11 mammalian PDE families have tandem ...GAF domains at their N termini. PDE10A may be the only mammalian PDE for which cAMP is the GAF domain ligand, and it may be allosterically stimulated by cAMP. PDE10A is highly expressed in striatal medium spiny neurons. Here we report the crystal structure of the C-terminal GAF domain (GAF-B) of human PDE10A complexed with cAMP at 2.1-Å resolution. The conformation of the PDE10A GAF-B domain monomer closely resembles those of the GAF domains of PDE2A and the cyanobacterium Anabaena cyaB2 adenylyl cyclase, except for the helical bundle consisting of α1, α2, and α5. The PDE10A GAF-B domain forms a dimer in the crystal and in solution. The dimerization is mainly mediated by hydrophobic interactions between the helical bundles in a parallel arrangement, with a large buried surface area. In the PDE10A GAF-B domain, cAMP tightly binds to a cNMP-binding pocket. The residues in the α3 and α4 helices, the β6 strand, the loop between 310 and α4, and the loop between α4 and β5 are involved in the recognition of the phosphate and ribose moieties. This recognition mode is similar to those of the GAF domains of PDE2A and cyaB2. In contrast, the adenine base is specifically recognized by the PDE10A GAF-B domain in a unique manner, through residues in the β1 and β2 strands.
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-ΔC) was solved in an oligomeric state. The molecular structure of BMRF1-ΔC 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-ΔC, 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-ΔC 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-ΔC associates with the catalytic subunit BALF5 around the dimer interface. Consequently, the monomeric form of BMRF1-ΔC probably binds to BALF5, because the steric consequences would prevent the maintenance of the dimeric form. A distinctive feature of BMRF1-ΔC is that the dimeric and monomeric forms might be utilized for the DNA binding and replication processes, respectively.
The nuclear pore complex mediates the transport of macromolecules across the nuclear envelope (NE). The vertebrate nuclear pore protein Nup35, the ortholog of
Saccharomyces cerevisiae Nup53p, is ...suggested to interact with the NE membrane and to be required for nuclear morphology. The highly conserved region between vertebrate Nup35 and yeast Nup53p is predicted to contain an RNA-recognition motif (RRM) domain. Due to its low level of sequence homology with other RRM domains, the RNP1 and RNP2 motifs have not been identified in its primary structure. In the present study, we solved the crystal structure of the RRM domain of mouse Nup35 at 2.7 Å resolution. The Nup35 RRM domain monomer adopts the characteristic βαββαβ topology, as in other reported RRM domains. The structure allowed us to locate the atypical RNP1 and RNP2 motifs. Among the RNP motif residues, those on the β-sheet surface are different from those of the canonical RRM domains, while those buried in the hydrophobic core are highly conserved. The RRM domain forms a homodimer in the crystal, in accordance with analytical ultracentrifugation experiments. The β-sheet surface of the RRM domain, with its atypical RNP motifs, contributes to homodimerization mainly by hydrophobic interactions: the side-chain of Met236 in the β4 strand of one Nup35 molecule is sandwiched by the aromatic side-chains of Phe178 in the β1 strand and Trp209 in the β3 strand of the other Nup35 molecule in the dimer. This structure reveals a new homodimerization mode of the RRM domain.
Rap2-interacting protein x (RPIPx) is a homolog of RPIP8, a specific effector of Rap2 GTPase. The N-terminal region of RPIP8,
which contains the RUN domain, interacts with Rap2. Using cell-free ...synthesis and NMR, we determined that the region encompassing
residues 83-255 of mouse RPIPx, which is 40-residues larger than the predicted RUN domain (residues 113-245), is the minimum
fragment that forms a correctly folded protein. This fragment, the RPIPx RUN domain, interacted specifically with Rap2B in vitro in a nucleotide-dependent manner. The crystal structure of the RPIPx RUN domain was determined at 2.0Ã of resolution by the
multiwavelength anomalous dispersion (MAD) method. The RPIPx RUN domain comprises eight anti-parallel α-helices, which form
an extensive hydrophobic core, followed by an extended segment. The residues in the core region are highly conserved, suggesting
the conservation of the RUN domain-fold among the RUN domain-containing proteins. The residues forming a positively charged
surface are conserved between RPIP8 and its homologs, suggesting that this surface is important for Rap2 binding. In the crystal
the putative Rap2 binding site of the RPIPx RUN domain interacts with the extended segment in a segment-swapping manner.
•Overexpression of the ZBR fragment of Emi2, but not of Emi1, induces abnormal cell division.•The Emi2 ZBR fragment impairs the association of the coactivator Cdc20 with APC/C.•The Emi2 ZBR fragment ...inhibits ubiquitylation by the cullin-RING of APC/C and E2C.•The Emi2 ZBR-specific residues for APC/C inhibitory activity have been identified.
Anaphase-promoting complex or cyclosome (APC/C) is a multisubunit ubiquitin ligase E3 that targets cell-cycle regulators. Cdc20 is required for full activation of APC/C in M phase, and mediates substrate recognition. In vertebrates, Emi2/Erp1/FBXO43 inhibits APC/C-Cdc20, and functions as a cytostatic factor that causes long-term M phase arrest of mature oocytes. In this study, we found that a fragment corresponding to the zinc-binding region (ZBR) domain of Emi2 inhibits cell-cycle progression, and impairs the association of Cdc20 with the APC/C core complex in HEK293T cells. Furthermore, we revealed that the ZBR fragment of Emi2 inhibits in vitro ubiquitin chain elongation catalyzed by the APC/C cullin-RING ligase module, the ANAPC2–ANAPC11 subcomplex, in combination with the ubiquitin chain-initiating E2, E2C/UBE2C/UbcH10. Structural analyses revealed that the Emi2 ZBR domain uses different faces for the two mechanisms. Thus, the double-faced ZBR domain of Emi2 antagonizes the APC/C function by inhibiting both the binding with the coactivator Cdc20 and ubiquitylation mediated by the cullin-RING ligase module and E2C. In addition, the tail region between the ZBR domain and the C-terminal RL residues the post-ZBR (PZ) region interacts with the cullin subunit, ANAPC2. In the case of the ZBR fragment of the somatic paralogue of Emi2, Emi1/FBXO5, these inhibitory activities against cell division and ubiquitylation were not observed. Finally, we identified two sets of key residues in the Emi2 ZBR domain that selectively exert each of the dual Emi2-specific modes of APC/C inhibition, by their mutation in the Emi2 ZBR domain and their transplantation into the Emi1 ZBR domain.
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