Kyoung-Seok Ryu

Structural insight into dimeric interaction of the SARAH domains from Mst1 and RASSF family proteins in the apoptosis pathway

In eukaryotic cells, apoptosis and cell cycle arrest by the Ras → RASSF → MST pathway are controlled by the interaction of SARAH (for Salvador/Rassf/Hippo) domains in the C-terminal part of tumor suppressor proteins. The Mst1 SARAH domain interacts with its homologous domain of Rassf1 and Rassf5 (also known as Nore1) by forming a heterodimer that mediates the apoptosis process. Here, we describe the homodimeric structure of the human Mst1 SARAH domain and its heterotypic interaction with the Rassf5 and Salvador (Sav) SARAH domain. The Mst1 SARAH structure forms a homodimer containing two helices per monomer. An antiparallel arrangement of the long α-helices (h2/h2′) provides an elongated binding interface between the two monomers, and the short 310 helices (h1/h1′) are folded toward that of the other monomer. Chemical shift perturbation experiments identified an elongated, tight-binding interface with the Rassf5 SARAH domain and a 1:1 heterodimer formation. The linker region between the kinase and the SARAH domain is shown to be disordered in the free protein. These results imply a novel mode of interaction with RASSF family proteins and provide insight into the mechanism of apoptosis control by the SARAH domain.

Structure of PP4397 Reveals the Molecular Basis for Different c-di-GMP Binding Modes by Pilz Domain Proteins.

Cyclic diguanylate (c-di-GMP) is a global regulator that modulates pathogen virulence and biofilm formation in bacteria. Although a bioinformatic study revealed that PilZ domain proteins are the long-sought c-di-GMP binding proteins, the mechanism by which c-di-GMP regulates them is uncertain. Pseudomonas putida PP4397 is one such protein that contains YcgR-N and PilZ domains and the apo-PP4397 structure was solved earlier by the Joint Center for Structural Genomics. We determined the crystal structure of holo-PP4397 and found that two intercalated c-di-GMPs fit into the junction of its YcgR-N and PilZ domains. Moreover, c-di-GMP binding induces PP4397 to undergo a dimer-to-monomer transition. Interestingly, another PilZ domain protein, VCA0042, binds to a single molecule of c-di-GMP, and both its apo and holo forms are dimeric. Mutational studies and the additional crystal structure of holo-VCA0042 (L135R) showed that the Arg122 residue of PP4397 is crucial for the recognition of two molecules of c-di-GMP. Thus, PilZ domain proteins exhibit different c-di-GMP binding stoichiometry and quaternary structure, and these differences are expected to play a role in generating diverse forms of c-di-GMP-mediated regulation.

Mechanism of anchoring of OmpA protein to the cell wall peptidoglycan of the gram-negative bacterial outer membrane

The outer membrane protein A (OmpA) plays important roles in anchoring of the outer membrane to the bacterial cell wall. The C‐terminal periplasmic domain of OmpA (OmpA‐like domain) associates with the peptidoglycan (PGN) layer noncovalently. However, there is a paucity of information on the structural aspects of the mechanism of PGN recognition by OmpA‐like domains. To elucidate this molecular recognition process, we solved the high‐resolution crystal structure of an OmpA‐like domain from Acinetobacter baumannii bound to diaminopimelate (DAP), a unique bacterial amino acid from the PGN. The structure clearly illustrates that two absolutely conserved Asp271 and Arg286 residues are the key to the binding to DAP of PGN. Identification of DAP as the central anchoring site of PGN to OmpA is further supported by isothermal titration calorimetry and a pulldown assay with PGN. An NMR‐based computational model for complexation between the PGN and OmpA emerged, and this model is validated by determining the crystal structure in complex with a synthetic PGN fragment. These structural data provide a detailed glimpse of how the anchoring of OmpA to the cell wall of gram‐negative bacteria takes place in a DAP‐dependent manner.—Park, J. S., Lee, W. C., Yeo, K. J., Ryu, K.‐S., Kumarasiri, M., Hesek, D., Lee, M., Mobashery, S., Song, J. H., Lim, S. I., Lee, J. C., Cheong, C., Jeon, Y. H., Kim, H.‐Y. Mechanism of anchoring of OmpA protein to the cell wall peptidoglycan of the gram‐negative bacterial outer membrane. FASEB J. 26, 219–228 (2012). www.fasebj.org

Structure of the Cdt1 C-terminal domain: Conservation of the winged helix fold in replication licensing factors

In eukaryotic replication licensing, Cdt1 plays a key role by recruiting the MCM2‐7 complex onto the origin of chromosome. The C‐terminal domain of mouse Cdt1 (mCdt1C), the most conserved region in Cdt1, is essential for licensing and directly interacts with the MCM2‐7 complex. We have determined the structures of mCdt1CS (mCdt1C_small; residues 452 to 557) and mCdt1CL (mCdt1C_large; residues 420 to 557) using X‐ray crystallography and solution NMR spectroscopy, respectively. While the N‐terminal 31 residues of mCdt1CL form a flexible loop with a short helix near the middle, the rest of mCdt1C folds into a winged helix structure. Together with the middle domain of mouse Cdt1 (mCdt1M, residues 172–368), this study reveals that Cdt1 is formed with a tandem repeat of the winged helix domain. The winged helix fold is also conserved in other licensing factors including archaeal ORC and Cdc6, which supports an idea that these replication initiators may have evolved from a common ancestor. Based on the structure of mCdt1C, in conjunction with the biochemical analysis, we propose a binding site for the MCM complex within the mCdt1C.

Codon optimization enhances protein expression of human peptide deformylase in E. coli

Human peptide deformylase (hPDF), located in the mitochondria, has recently become a promising target for anti-cancer therapy. However, the expression of the hPDF gene in Escherichia coli is not efficient likely due to extremely high levels of GC content as well as the presence of rare codons. We performed codon optimization of the hPDF gene in order to reduce GC content and to eliminate rare codons. Putative stable secondary structures of the optimized gene were also reduced. Codon optimization increased the expression of hPDF protein (residues 63-243) presumably by reducing the GC content. A large amount of soluble hPDF was obtained upon its fusion with thioredoxin (Trx-hPDF), although an insoluble fraction was still dominant. We confirmed that Co(2+) is an optimal metal for increasing the activity of purified Trx-hPDF, and that actinonin acts as an efficient inhibitor. Therefore, a large amount of purified hPDF protein would provide many benefits for the screening of various drug candidates.

Structural characterization reveals that a PilZ domain protein undergoes substantial conformational change upon binding to cyclic dimeric guanosine monophosphate

PA4608 is a single PilZ domain protein from Pseudomonas aeruginosa that binds to cyclic dimeric guanosine monophosphate (c‐di‐GMP). Although the monomeric structure of unbound PA4608 has been studied in detail, the molecular details of c‐di‐GMP binding to this protein are still uncharacterized. Hence, we determined the solution structure of c‐di‐GMP bound PA4608. We found that PA4608 undergoes conformational changes to expose the c‐di‐GMP binding site by ejection of the C‐terminal 310 helix. A dislocation of the C‐terminal tail in the presence of c‐di‐GMP implies that this region acts as a lid that alternately covers and exposes the hydrophobic surface of the binding site. In addition, mutagenesis and NOE data for PA4608 revealed that conserved residues are in contact with the c‐di‐GMP molecule. The unique structural characteristics of PA4608, including its monomeric state and its ligand binding characteristics, yield insight into its function as a c‐di‐GMP receptor.

Structural insights into the dual-targeting mechanism of Nutlin-3.

Multi-targeting therapy is an emerging strategy of drug discovery to improve therapeutic efficacy, safety and resistance profiles. In this study, we monitored the binding of a potent MDM2 inhibitor Nutlin-3 with anti-apoptotic Bcl-2 family proteins using NMR spectroscopy. Our results showed the universal binding of Nutlin-3 with diverse anti-apoptotic Bcl-2 family proteins. Taken together with the binding data for Nutlin-3 analogs, the structural model of the Bcl-X(L)/Nutlin-3 complex showed that the binding mode of Nutlin-3 resembles that of the Bcl-X(L)/Bcl-2 inhibitors, suggesting the molecular mechanism of transcription-independent mitochondrial apoptosis by Nutlin-3. Finally, our structural comparison provides structural insights into the dual-targeting mechanism of how Nutlin-3 can bind to two different target proteins, MDM2 and anti-apoptotic Bcl-2 family proteins in a similar manner.

The Human Cdc34 Carboxyl Terminus Contains a Non-covalent Ubiquitin Binding Activity That Contributes to SCF-dependent Ubiquitination

Cdc34 is an E2 ubiquitin-conjugating enzyme that functions in conjunction with SCF (Skp1·Cullin 1·F-box) E3 ubiquitin ligase to catalyze covalent attachment of polyubiquitin chains to a target protein. Here we identified direct interactions between the human Cdc34 C terminus and ubiquitin using NMR chemical shift perturbation assays. The ubiquitin binding activity was mapped to two separate Cdc34 C-terminal motifs (UBS1 and UBS2) that comprise residues 206–215 and 216–225, respectively. UBS1 and UBS2 bind to ubiquitin in the proximity of ubiquitin Lys48 and C-terminal tail, both of which are key sites for conjugation. When bound to ubiquitin in one orientation, the Cdc34 UBS1 aromatic residues (Phe206, Tyr207, Tyr210, and Tyr211) are probably positioned in the vicinity of ubiquitin C-terminal residue Val70. Replacement of UBS1 aromatic residues by glycine or of ubiquitin Val70 by alanine decreased UBS1-ubiquitin affinity interactions. UBS1 appeared to support the function of Cdc34 in vivo because human Cdc34(1–215) but not Cdc34(1–200) was able to complement the growth defect by yeast Cdc34 mutant strain. Finally, reconstituted IκBα ubiquitination analysis revealed a role for each adjacent pair of UBS1 aromatic residues (Phe206/Tyr207, Tyr210/Tyr211) in conjugation, with Tyr210 exhibiting the most pronounced catalytic function. Intriguingly, Cdc34 Tyr210 was required for the transfer of the donor ubiquitin to a receptor lysine on either IκBα or a ubiquitin in a manner that depended on the neddylated RING sub-complex of the SCF. Taken together, our results identified a new ubiquitin binding activity within the human Cdc34 C terminus that contributes to SCF-dependent ubiquitination.

Structure and interaction of ubiquitin-associated domain of human Fas-associated factor 1.

Fas‐associated factor (FAF)‐1 is a multidomain protein that was first identified as a member of the Fas death‐inducing signaling complex, but later found to be involved in various biological processes. Although the exact mechanisms are not clear, FAF1 seems to play an important role in cancer, asbestos‐induced mesotheliomas, and Parkinsons disease. It interacts with polyubiquitinated proteins, Hsp70, and p97/VCP (valosin‐containing protein), in addition to the proteins of the Fas‐signaling pathway. We have determined the crystal structure of the ubiquitin‐associated domain of human FAF1 (hFAF1‐UBA) and examined its interaction with ubiquitin and ubiquitin‐like proteins using nuclear magnetic resonance. hFAF1‐UBA revealed a canonical three‐helical bundle that selectively binds to mono‐ and di‐ubiquitin (Lys48‐linked), but not to SUMO‐1 (small ubiquitin‐related modifier 1) or NEDD8 (neural precursor cell expressed, developmentally down‐regulated 8). The interaction between hFAF1‐UBA and di‐ubiquitin involves hydrophobic interaction accompanied by a transition in the di‐ubiquitin conformation. These results provide structural insight into the mechanism of polyubiquitin recognition by hFAF1‐UBA.

Biotinoyl domain of human acetyl-CoA carboxylase: Structural insights into the carboxyl transfer mechanism.

Acetyl‐CoA carboxylase (ACC) catalyzes the first step in fatty acid biosynthesis: the synthesis of malonyl‐CoA from acetyl‐CoA. As essential regulators of fatty acid biosynthesis and metabolism, ACCs are regarded as therapeutic targets for the treatment of metabolic diseases such as obesity. In ACC, the biotinoyl domain performs a critical function by transferring an activated carboxyl group from the biotin carboxylase domain to the carboxyl transferase domain, followed by carboxyl transfer to malonyl‐CoA. Despite the intensive research on this enzyme, only the bacterial and yeast ACC structures are currently available. To explore the mechanism of ACC holoenzyme function, we determined the structure of the biotinoyl domain of human ACC2 and analyzed its characteristics and interaction with the biotin ligase, BirA using NMR spectroscopy. The 3D structure of the hACC2 biotinoyl domain has a similar folding topology to the earlier determined domains from E. coli and P. shermanii. However, the local structures near the biotinylation sites have notable differences that include the geometry of the consensus “Met‐Lys‐Met” (MKM) motif and the absence of “thumb” structure in the hACC2 biotinoyl domain. Observations of the NMR signals upon the biotinylation indicate that the biotin group of hACC2 does not affect the structure of the biotinoyl domain, while the biotin group for E. coli ACC interacts directly with the thumb residues that are not present in the hACC2 structure. These results imply that, in the E. coli ACC reaction, the biotin moiety carrying the carboxyl group from BC to CT can pause at the thumb of the BCCP domain. The human biotinoyl domain, however, lacks the thumb structure and does not have additional noncovalent interactions with the biotin moiety; thus, the flexible motion of the biotinylated lysine residue must underlie the “swinging arm” motion. The chemical shift perturbation and the cross saturation experiments of the human ACC2 holo‐biotinoyl upon the addition of the biotin ligase (BirA) showed the interaction surface near the MKM motif, the two glutamic acids (Glu 926, Glu 953), and the positively charged residues (several lysine and arginine residues). This study provides insight into the mechanism of ACC holoenzyme function and supports the swinging arm model in human ACCs. Proteins 2008.