Eunice EunKyeong Kim

Crystal Structures of Enoyl-ACP Reductases I (FabI) and III (FabL) from B. subtilis

Enoyl-[acyl carrier protein] (ACP) reductase (ENR) is a key enzyme in type II fatty acid synthesis that catalyzes the last step in each elongation cycle. Therefore, it has been considered as a target for antibiotics. However, recent studies indicate that some pathogens have more than one ENR; in particular, Bacillus subtilis has two ENRs, FabI and FabL. The crystal structures of the ternary complexes of BsFaBI and BsFabL are found as a homotetramer showing the same overall structure despite a sequence identity of only 24%. The positions of the catalytic dyad of Tyr-(Xaa)(6)-Lys in FabL are almost identical to that of FabI, but a detailed structural analysis shows that FabL shares more structural similarities with FabG and other members of the SDR (short-chain alcohol dehydrogenase/reductase) family. The apo FabL structure shows significantly different conformations at the cofactor and the substrate-binding regions, and this resulted in a totally different tetrameric arrangement reflecting the flexibility of these regions in the absence of the cofactor and substrate/inhibitor.

Structural Basis for Ufm1 Processing by UfSP1

Ubiquitin-fold modifier 1 (Ufm1) is a newly identified ubiquitin-like protein. Like ubiquitin and other ubiquitin-like proteins, Ufm1 is synthesized as a precursor that needs to be processed to expose the conserved C-terminal glycine prior to its conjugation to target proteins. Two novel proteases, named UfSP1 and UfSP2, have been shown to be responsible for the release of Ufm1 from Ufm1-conjugated cellular proteins as well as for the processing of its precursor. They show no sequence homology with known proteases. Here, we describe the 1.7Å resolution crystal structure of mouse UfSP1, consisting of 217 amino acids. The structure reveals that it is a novel cysteine protease having a papain-like fold, with Cys53, Asp175, and His177 that form a catalytic triad, and Tyr41 that participates in the formation of the oxyanion hole. This differs from the canonical catalytic triad of papain-like proteases in that the aspartate and the histidine residues are from the “Asp-Pro-His” box. The Asp-Pro-His configuration seen in UfSP1, together with Atg4B and M48USP, seem to form a new subfamily of the cysteine protease superfamily. The mutagenesis study of the active site residues confirms structural basis for catalysis. The interaction between UfSP1 and Ufm1 appears quite substantial, since the KD value was estimated to be 1.6 μm by the isothermal titration calorimetry analysis. Furthermore, the NMR data shows that the loop between β3 and α2 in addition to the C-terminal region of Ufm1 plays a role in binding to UfSP1.

Structural insights into Staphylococcus aureus enoyl-ACP reductase (FabI), in complex with NADP and triclosan.

Structural insights into Staphylococcus aureus enoyl-ACP reductase (FabI), in complex with NADP and triclosan Amit Priyadarshi, Eunice EunKyeong Kim,* and Kwang Yeon Hwang* 1Division of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 136-701, South Korea 2 Biomedical Research Center, Korea Institute of Science and Technology, Seoul 136-791, South Korea 3 Biomolecular Science, University of Science and Technology, Yuseong-gu, Daejeon 305-333, South Korea

Structure of Ubiquitin-fold Modifier 1-specific Protease UfSP2

Ubiquitin-fold modifier 1 (Ufm1)-specific protease 2 (UfSP2) is a cysteine protease that is responsible for the release of Ufm1 from Ufm1-conjugated cellular proteins, as well as for the generation of mature Ufm1 from its precursor. The 2.6 Å resolution crystal structure of mouse UfSP2 reveals that it is composed of two domains. The C-terminal catalytic domain is similar to UfSP1 with Cys294, Asp418, His420, Tyr282, and a regulatory loop participating in catalysis. The novel N-terminal domain shows a unique structure and plays a role in the recognition of its cellular substrate C20orf116 and thus in the recruitment of UfSP2 to the endoplasmic reticulum, where C20orf116 predominantly localizes. Mutagenesis studies were carried out to provide the structural basis for understanding the loss of catalytic activity observed in a recently identified UfSP2 mutation that is associated with an autosomal dominant form of hip dysplasia.

Structural Basis for Ovarian Tumor Domain-containing Protein 1 (OTU1) Binding to p97/Valosin-containing Protein (VCP)

Background: Ovarian tumor domain-containing protein 1 (OTU1) acts as a deubiquitinating enzyme in the endoplasmic reticulum-associated degradation (ERAD) pathway by associating with valosin-containing protein (VCP). Results: One OTU1 binds to a VCP hexamer with a KD of 0.71 μm. Conclusion: The 39GYPP42 (S3/S4) loop of OTU1 is critical for interaction with VCP. Significance: This is the first structural study of VCP and OTU1 complex. Valosin-containing protein (VCP), also known as p97, is an AAA+ ATPase that plays an essential role in a broad array of cellular processes including the endoplasmic reticulum-associated degradation (ERAD) pathway. Recently, ERAD-specific deubiquitinating enzymes have been reported to be physically associated with VCP, although the exact mechanism is not yet clear. Among these enzymes is ovarian tumor domain-containing protein 1 (OTU1). Here, we report the structural basis for interaction between VCP and OTU1. The crystal structure of the ubiquitin regulatory X-like (UBXL) domain of OTU1 (UBXLOTU1) complexed to the N-terminal domain of VCP (NVCP) at 1.8-Å resolution reveals that UBXLOTU1 adopts a ubiquitin-like fold and binds at the interface of two subdomains of NVCP using the 39GYPP42 loop of UBXLOTU1 with the two prolines in cis- and trans-configurations, respectively. A mutagenesis study shows that this loop is not only critical for the interaction with VCP but also for its role in the ERAD pathway. Negative staining EM shows that one molecule of OTU1 binds to one VCP hexamer, and isothermal titration calorimetry suggests that the two proteins bind with a KD of 0.71 μm. Analytical size exclusion chromatography and isothermal titration calorimetry demonstrates that OTU1 can bind VCP in both the presence and absence of a heterodimer formed by ubiquitin fusion degradation protein 1 and nuclear localization protein 4.

New design platform for malonyl-CoA-acyl carrier protein transacylase

Malonyl‐CoA‐acyl carrier protein transacylase (MCAT) transfers the malonyl group from malonyl‐CoA to holo‐acyl carrier protein (ACP), and since malonyl‐ACP is a key building block for fatty‐acid biosynthesis it is considered as a promising antibacterial target. The crystal structures of MCAT from Staphylococcus aureus and Streptococcus pneumoniae have been determined at 1.46 and 2.1 Å resolution, respectively. In the SaMCAT structure, the N‐terminal expression peptide of a neighboring molecule running in the opposite direction of malonyl‐CoA makes extensive interactions with the highly conserved “Gly‐Gln‐Gly‐Ser‐Gln” stretch, suggesting a new design platform. Mutagenesis results suggest that Ser91 and His199 are the catalytic dyad.

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.

Structural and functional insights into the regulation mechanism of CK2 by IP6 and the intrinsically disordered protein Nopp140

Significance Structural and functional studies on protein kinase CK2α, which is a ubiquitous kinase that can phosphorylate hundreds of cellular proteins, revealed that CK2α activity is inhibited by Nopp140 and reactivated by IP6 by competitive binding at the substrate recognition site of CK2α. IP6 binds to the lysine-rich cluster of CK2α, and phospho-Ser574 on Nopp140 significantly enhances its interaction with CK2α. Protein kinase CK2 is a ubiquitous kinase that can phosphorylate hundreds of cellular proteins and plays important roles in cell growth and development. Deregulation of CK2 is related to a variety of human cancers, and CK2 is regarded as a suppressor of apoptosis; therefore, it is a target of anticancer therapy. Nucleolar phosphoprotein 140 (Nopp140), which is an intrinsically disordered protein, interacts with CK2 and inhibits the latter’s catalytic activity in vitro. Interestingly, the catalytic activity of CK2 is recovered in the presence of d-myo-inositol 1,2,3,4,5,6-hexakisphosphate (IP6). IP6 is widely distributed in animal cells, but the molecular mechanisms that govern its cellular functions in animal cells have not been completely elucidated. In this study, the crystal structure of CK2 in complex with IP6 showed that the lysine-rich cluster of CK2 plays an important role in binding to IP6. The biochemical experiments revealed that a Nopp140 fragment (residues 568–596) and IP6 competitively bind to the catalytic subunit of CK2 (CK2α), and phospho-Ser574 of Nopp140 significantly enhances its interaction with CK2α. Substitutions of K74E, K76E, and K77E in CK2α significantly reduced the interactions of CK2α with both IP6 and the Nopp140-derived peptide. Our study gives an insight into the regulation of CK2. In particular, our work suggests that CK2 activity is inhibited by Nopp140 and reactivated by IP6 by competitive binding at the substrate recognition site of CK2.

Structural basis for recruiting and shuttling of the spliceosomal deubiquitinase USP4 by SART3

Abstract Squamous cell carcinoma antigen recognized by T-cells 3 (SART3) is a U4/U6 recycling factor as well as a targeting factor of USP4 and USP15. However, the details of how SART3 recognizes these deubiquitinases and how they get subsequently translocated into the nucleus are not known. Here, we present the crystal structures of the SART3 half-a-tetratricopeptide (HAT) repeat domain alone and in complex with the domain present in ubiquitin-specific protease (DUSP)-ubiquitin-like (UBL) domains of ubiquitin specific protease 4 (USP4). The 12 HAT repeats of SART3 are in two sub-domains (HAT-N and HAT-C) forming a dimer through HAT-C. USP4 binds SART3 at the opposite surface of the HAT-C dimer interface utilizing the β-structured linker between the DUSP and the UBL domains. The binding affinities of USP4 and USP15 to SART3 are 0.9 μM and 0.2 μM, respectively. The complex structure of SART3 nuclear localization signal (NLS) and importin-α reveals bipartite binding, and removal of SART3 NLS prevents the entry of USP4 (and USP15) into the nucleus and abrogates the subsequent deubiquitinase activity of USP4.

Structural insights into mouse anti-apoptotic Bcl-xl reveal affinity for Beclin 1 and gossypol.

This study reports the crystal structures of Bcl-xl wild type and three Bcl-xl mutants (Y101A, F105A, and R139A) with amino acid substitutions in the hydrophobic groove of the Bcl-xl BH3 domain. An additional 12 ordered residues were observed in a highly flexible loop between the alpha1 and alpha2 helices, and were recognized as an important deamidation site for the regulation of apoptosis. The autophagy-effector protein, Beclin 1, contains a novel BH3 domain (residues 101-125), which binds to the surface cleft of Bcl-xl, as confirmed by nuclear magnetic resonance (NMR) spectroscopy and analytical gel-filtration results. Gossypol, a potent inhibitor of Bcl-xl, had a K(d) value of 0.9 microM. In addition, the structural and biochemical analysis of five Bcl-xl substitution mutants will provide structural insights into the design and development of anti-cancer drugs.