Jeong-Yong Suh

Visualizing transient events in amino-terminal autoprocessing of HIV-1 protease.

HIV-1 protease processes the Gag and Gag-Pol polyproteins into mature structural and functional proteins, including itself, and is therefore indispensable for viral maturation. The mature protease is active only as a dimer with each subunit contributing catalytic residues. The full-length transframe region protease precursor appears to be monomeric yet undergoes maturation via intramolecular cleavage of a putative precursor dimer, concomitant with the appearance of mature-like catalytic activity. How such intramolecular cleavage can occur when the amino and carboxy termini of the mature protease are part of an intersubunit β-sheet located distal from the active site is unclear. Here we visualize the early events in N-terminal autoprocessing using an inactive mini-precursor with a four-residue N-terminal extension that mimics the transframe region protease precursor. Using paramagnetic relaxation enhancement, a technique that is exquisitely sensitive to the presence of minor species, we show that the mini-precursor forms highly transient, lowly populated (3–5%) dimeric encounter complexes that involve the mature dimer interface but occupy a wide range of subunit orientations relative to the mature dimer. Furthermore, the occupancy of the mature dimer configuration constitutes a very small fraction of the self-associated species (accounting for the very low enzymatic activity of the protease precursor), and the N-terminal extension makes transient intra- and intersubunit contacts with the substrate binding site and is therefore available for autocleavage when the correct dimer orientation is sampled within the encounter complex ensemble.

Solution Structure of the 128 kDa Enzyme I Dimer from Escherichia coli and Its 146 kDa Complex with HPr Using Residual Dipolar Couplings and Small- and Wide-Angle X-ray Scattering

The solution structures of free Enzyme I (EI, ∼128 kDa, 575 × 2 residues), the first enzyme in the bacterial phosphotransferase system, and its complex with HPr (∼146 kDa) have been solved using novel methodology that makes use of prior structural knowledge (namely, the structures of the dimeric EIC domain and the isolated EIN domain both free and complexed to HPr), combined with residual dipolar coupling (RDC), small- (SAXS) and wide- (WAXS) angle X-ray scattering and small-angle neutron scattering (SANS) data. The calculational strategy employs conjoined rigid body/torsion/Cartesian simulated annealing, and incorporates improvements in calculating and refining against SAXS/WAXS data that take into account complex molecular shapes in the description of the solvent layer resulting in a better representation of the SAXS/WAXS data. The RDC data orient the symmetrically related EIN domains relative to the C(2) symmetry axis of the EIC dimer, while translational, shape, and size information is provided by SAXS/WAXS. The resulting structures are independently validated by SANS. Comparison of the structures of the free EI and the EI-HPr complex with that of the crystal structure of a trapped phosphorylated EI intermediate reveals large (∼70-90°) hinge body rotations of the two subdomains comprising the EIN domain, as well as of the EIN domain relative to the dimeric EIC domain. These large-scale interdomain motions shed light on the structural transitions that accompany the catalytic cycle of EI.

Mechanistic details of a protein-protein association pathway revealed by paramagnetic relaxation enhancement titration measurements

Protein–protein association generally proceeds via the intermediary of a transient, lowly populated, encounter complex ensemble. The mechanism whereby the interacting molecules in this ensemble locate their final stereospecific structure is poorly understood. Further, a fundamental question is whether the encounter complex ensemble is an effectively homogeneous population of nonspecific complexes or whether it comprises a set of distinct structural and thermodynamic states. Here we use intermolecular paramagnetic relaxation enhancement (PRE), a technique that is exquisitely sensitive to lowly populated states in the fast exchange regime, to characterize the mechanistic details of the transient encounter complex interactions between the N-terminal domain of Enzyme I (EIN) and the histidine-containing phosphocarrier protein (HPr), two major bacterial signaling proteins. Experiments were conducted at an ionic strength of 150 mM NaCl to eliminate any spurious nonspecific associations not relevant under physiological conditions. By monitoring the dependence of the intermolecular transverse PRE (Γ2) rates measured on 15N-labeled EIN on the concentration of paramagnetically labeled HPr, two distinct types of encounter complex configurations along the association pathway are identified and dissected. The first class, which is in equilibrium with and sterically occluded by the specific complex, probably involves rigid body rotations and small translations near or at the active site. In contrast, the second class of encounter complex configurations can coexist with the specific complex to form a ternary complex ensemble, which may help EIN compete with other HPr binding partners in vivo by increasing the effective local concentration of HPr even when the active site of EIN is occupied.

Molecular determinants of ovarian cancer chemoresistance: new insights into an old conundrum.

Ovarian cancer is the most lethal gynecological malignancy. Cisplatin and its derivatives are first‐line chemotherapeutics, and their resistance is a major hurdle in successful ovarian cancer treatment. Understanding the molecular dysregulation underlying chemoresistance is important for enhancing therapeutic outcome. Here, we review two established pathways in cancer chemoresistance. p53 is a major tumor suppressor regulating proliferation and apoptosis, and its mutation is a frequent event in human malignancies. The PI3K/Akt axis is a key oncogenic pathway regulating survival and tumorigenesis by controlling several tumor suppressors, including p53. The interplay between these pathways is well established, although the oncogenic phosphatase PPM1D adds a new layer to this intricate relationship and provides new insights into the processes determining cell fate. Inhibition of the PI3K/Akt pathway by functional food compounds as an adjunct to chemotherapeutics may tip the balance in favor of apoptosis rather than survival, enhancing therapeutic efficacy, and reducing side effects.

Solution NMR Structure of the Barrier-to-Autointegration Factor-Emerin Complex

The barrier-to-autointegration factor BAF binds to the LEM domain (EmLEM) of the nuclear envelope protein emerin and plays an essential role in the nuclear architecture of metazoan cells. In addition, the BAF2 dimer bridges and compacts double-stranded DNA nonspecifically via two symmetry-related DNA binding sites. In this article we present biophysical and structural studies on a complex of BAF2 and EmLEM. Light scattering, analytical ultracentrifugation, and NMR indicate a stoichiometry of one molecule of EmLEM bound per BAF2 dimer. The equilibrium dissociation constant (Kd) for the interaction of the BAF2 dimer and EmLEM, determined by isothermal titration calorimetry, is 0.59 ± 0.03 μm. Z-exchange spectroscopy between corresponding cross-peaks of the magnetically non-equivalent subunits of the BAF2 dimer in the complex yields a dissociation rate constant of 78 ± 2s-1. The solution NMR structure of the BAF2-EmLEM complex reveals that the LEM and DNA binding sites on BAF2 are non-overlapping and that both subunits of the BAF2 dimer contribute approximately equally to the EmLEM binding site. The relevance of the implications of the structural and biophysical data on the complex in the context of the interaction between the BAF2 dimer and EmLEM at the nuclear envelope is discussed.

Piceatannol Enhances Cisplatin Sensitivity in Ovarian Cancer via Modulation of p53, X-linked Inhibitor of Apoptosis Protein (XIAP), and Mitochondrial Fission

Background: Chemotherapeutic sensitivity in ovarian cancer is dependent on effective apoptosis signaling. Results: Piceatannol enhances cisplatin sensitivity by modulating p53, XIAP (X-linked inhibitor of apoptosis protein), and mitochondrial fission in vitro and in vivo. Conclusion: Piceatannol is a potent enhancer of cisplatin-induced apoptosis. Significance: Piceatannol exhibits potential for clinical development for the treatment of ovarian cancer. Resistance to cisplatin (CDDP) in ovarian cancer (OVCA) arises from the dysregulation of tumor suppressors and survival signals. During genotoxic challenge, these factors can be influenced by secondary agents that facilitate the induction of apoptosis. Piceatannol is a natural metabolite of the stilbene resveratrol found in grapes and is converted from its parent compound by the enzyme CYP1BA1 p450. It has been hypothesized to exert specific effects against various cellular targets; however, its ability to influence CDDP resistance in cancer cells has not been investigated to date. Here, we show that piceatannol is a potent enhancer of CDDP sensitivity in OVCA, and this effect is achieved through the modulation of several major determinants of chemoresistance. Piceatannol enhances p53-mediated expression of the pro-apoptotic protein NOXA, increases XIAP degradation via the ubiquitin-proteasome pathway, and enhances caspase-3 activation. This response is associated with an increase in Drp1-dependent mitochondrial fission, leading to more effective induction of apoptosis. In vivo studies using a mouse model of OVCA reveal that a number of these changes occur in association with a greater overall reduction in tumor weight when mice are treated with both piceatannol and CDDP, in comparison to treatment with either agent alone. Taken together, these findings demonstrate the potential application of piceatannol to enhance CDDP sensitivity in OVCA, and it acts on p53, XIAP, and mitochondrial fission.

Unusually stable helical kink in the antimicrobial peptide — A derivative of gaegurin

The structure of an active analog of the antibacterial peptide gaegurin was investigated by CD and NMR spectroscopy. The NOE connectivities showed that 21 out of 24 residues formed an α‐helix despite the presence of a central proline. CD and NMR analysis indicates that the helix is in fast equilibrium with random coil. From chemical shift analysis of the amide protons, the distances of hydrogen bonding in the helix were calculated, and manifested obvious periodicity which implied a kink in the middle of the helix. 1D amide proton exchange experiments provided further evidence of an exceptionally stable kink. It is inferred that this kink is important not only to the function of the peptide but also to the early stage of the folding as a nucleation site.

Structural basis for the auxin-induced transcriptional regulation by Aux/IAA17

Significance Auxin is the central hormone that governs diverse developmental processes in plants. Auxin response is regulated by auxin response transcription factor (ARF) and Aux/IAA transcriptional repressor. ARF and Aux/IAA form homo-oligomers and also hetero-oligomers for transcriptional regulation of auxin-response genes. Mechanistic understanding of how ARF and Aux/IAA change their association states is not well established. This work reports, to our knowledge, the first structure of the oligomerization domain of IAA17, and describes the key determinant that dictates the switch between homo- and hetero-oligomers. While Aux/IAA and ARF use a common scaffold and interface for homotypic and heterotypic associations, the charge composition at the interface determines the affinity and the oligomerization states. Based on the results, we propose a refined model of auxin-induced transcriptional regulation. Auxin is the central hormone that regulates plant growth and organ development. Transcriptional regulation by auxin is mediated by the auxin response factor (ARF) and the repressor, AUX/IAA. Aux/IAA associates with ARF via domain III−IV for transcriptional repression that is reversed by auxin-induced Aux/IAA degradation. It has been known that Aux/IAA and ARF form homo- and hetero-oligomers for the transcriptional regulation, but what determines their association states is poorly understood. Here we report, to our knowledge, the first solution structure of domain III−IV of Aux/IAA17 (IAA17), and characterize molecular interactions underlying the homotypic and heterotypic oligomerization. The structure exhibits a compact β-grasp fold with a highly dynamic insert helix that is unique in Aux/IAA family proteins. IAA17 associates to form a heterogeneous ensemble of front-to-back oligomers in a concentration-dependent manner. IAA17 and ARF5 associate to form homo- or hetero-oligomers using a common scaffold and binding interfaces, but their affinities vary significantly. The equilibrium dissociation constants (KD) for homo-oligomerization are 6.6 μM and 0.87 μM for IAA17 and ARF5, respectively, whereas hetero-oligomerization reveals a ∼10- to ∼100-fold greater affinity (KD = 73 nM). Thus, individual homo-oligomers of IAA17 and ARF5 spontaneously exchange their subunits to form alternating hetero-oligomers for transcriptional repression. Oligomerization is mainly driven by electrostatic interactions, so that charge complementarity at the interface determines the binding affinity. Variable binding affinity by surface charge modulation may effectively regulate the complex interaction network between Aux/IAA and ARF family proteins required for the transcriptional control of auxin-response genes.

Bio‐Inspired Design and Potential Biomedical Applications of a Novel Class of High‐Affinity Peptides

Antibodies have been widely used in a range of biopharmaceutical and biomedical applications due to their intrinsic high affinity and specificity toward various targets. However, poor tissue penetration owing to their large size, undesired effector functions, immunogenicity, costly recombinant production in mammalian cells, and complex intellectual property barriers (royalty stacking) have led researchers to seek alternatives to antibodies. Protein-scaffold-based affinity molecules and oligo DNA or RNA-based aptamers have recently emerged as novel high-affinity molecules that have indeed demonstrated potential utility in diagnosis and therapy. A common feature of such high-affinity molecules is that they possess three-dimensional folded structures that facilitate target binding through a large recognition interface, resulting in tight target binding with high specificity. Highaffinity molecules with small molecular mass exhibit rapid extravasation and higher tissue penetration than bigger highaffinity molecules and can be used as cancer diagnostics or therapeutics. To date, however, few reports have described the development of peptide-scaffold-based affinity molecules, presumably because it is difficult for peptides to form robust pre-organized structures. The knottin family and phylomers are peptide scaffolds engineered from naturally occurring protein domains; tight peptide binders with micromolar or nanomolar affinities can be selected from these two families. However, multiple disulfide bonds are involved in stabilizing knottin scaffolds. Thus, structural determination is necessary to ensure assumption of the correct fold, which limits the speed of product development. Phylomers are not peptide scaffolds with a defined, single structural framework; they rather consist of a library of diverse naturally occurring structural frameworks derived from foreign sources. Therefore, the structures of selected hits must be solved to figure out the specific scaffold, and there is a concern on potential immunogenicity because of the origin of phylomers. To our knowledge, no reports have described artificial peptide scaffolds that can be used as a general source of highaffinity peptides and that exhibit affinities toward a variety of biological targets in the nanomolar range. An artificial peptide scaffold must satisfy the following design criteria: 1) it should be able to form a secondary structure; 2) its preorganized structure should be stable and robust; 3) it should have a sufficient number of variable (randomizable) amino acids to create diversity; and 4) its variable regions should minimally affect its secondary structure. Herein, inspired by the structure of basic leucine zipper (bZIP) proteins, which function as transcriptional regulators in all eukaryotes through high-affinity (ca. low nanomolar), sequence-specific recognition of unique DNA motifs, we rationally designed novel artificial peptide-scaffold-based affinity molecules that form robust pre-organized structures and are capable of binding targets with high affinity and specificity. Homoor heterodimeric bZIP proteins have a basic region that abuts a sequence of hepta-leucine repeats (Figure 1a). The upper leucine-zipper region serves as a scaffold that maintains a unique open-mouthed structure through which variable basic regions are used to recognize DNA sites. Exploiting these features, we designed new artificial high-affinity peptide ligands, which we have termed “aptides”, from aptamer-like peptides. An aptide comprises a stabilizing scaffold and two target-binding regions (Figure 1a). The scaffold consists of a small (12 amino acids) but highly stable tryptophan zipper (trpzip; Tm = 72 8C) that forms a leucine-zipper-like b-hairpin structure, in which two tryptophan–tryptophan cross-strand pairs create a robust and stable structure. To mimic the DNA recognition site of bZIP proteins two target-binding regions, each comprising six randomizable amino acids, are introduced at both ends of the trpzip scaffold through glycine linkers. We expected that aptides would bind their target molecules with high affinity and specificity as a result of the synergistic action of the two target-binding sites. We used phage display to explore the feasibility of using aptide libraries as a source of high-affinity peptide candidates for screening against protein targets. For functional selection, the aptide libraries were adapted for monovalent display on filamentous bacteriophage surfaces. As an initial target protein for evaluating the aptide platform, we chose human fibronectin extradomain B (EDB), which is a validated tumor-specific biomarker that has been used in tumor imaging and therapy. Phage-display-based selections of the aptide library (8 10) against biotinylated EDB bound to [*] S. Kim, D. Kim, H. H. Jung, I.-H. Lee, Prof. J. I. Kim, Prof. S. Jon School of Life Sciences Gwangju Institute of Science and Technology (GIST) 123 Cheomdangwagi-ro, Gwangju 500-712 (South Korea) E-mail: syjon@gist.ac.kr

Solution Structure of a Post-transition State Analog of the Phosphotransfer Reaction between the A and B Cytoplasmic Domains of the Mannitol Transporter IIMannitol of the Escherichia coli Phosphotransferase System

The solution structure of the post-transition state complex between the isolated cytoplasmic A (IIAMtl) and phosphorylated B (phospho-IIBMtl) domains of the mannitol transporter of the Escherichia coli phosphotransferase system has been solved by NMR. The active site His-554 of IIAMtl was mutated to glutamine to block phosphoryl transfer activity, and the active site Cys-384 of IIBMtl (residues of IIBMtl are denoted in italic type) was substituted by serine to permit the formation of a stable phosphorylated form of IIBMtl. The two complementary interaction surfaces are predominantly hydrophobic, and two methionines on IIBMtl, Met-388 and Met-393, serve as anchors by interacting with two deep pockets on the surface of IIAMtl. With the exception of a salt bridge between the conserved Arg-538 of IIAMtl and the phosphoryl group of phospho-IIBMtl, electrostatic interactions between the two proteins are limited to the outer edges of the interface, are few in number, and appear to be weak. This accounts for the low affinity of the complex (Kd ∼ 3.7 mm), which is optimally tuned to the intact biological system in which the A and B domains are expressed as a single polypeptide connected by a flexible 21-residue linker. The phosphoryl transition state can readily be modeled with no change in protein-protein orientation and minimal perturbations in both the backbone immediately adjacent to His-554 and Cys-384 and the side chains in close proximity to the phosphoryl group. Comparison with the previously solved structure of the IIAMtl-HPr complex reveals how IIAMtl uses the same interaction surface to recognize two structurally unrelated proteins and explains the much higher affinity of IIAMtl for HPr than IIBMtl