Sam-Yong Park

Structural basis for the selective inhibition of JNK1 by the scaffolding protein JIP1 and SP600125

The c‐jun N‐terminal kinase (JNK) signaling pathway is regulated by JNK‐interacting protein‐1 (JIP1), which is a scaffolding protein assembling the components of the JNK cascade. Overexpression of JIP1 deactivates the JNK pathway selectively by cytoplasmic retention of JNK and thereby inhibits gene expression mediated by JNK, which occurs in the nucleus. Here, we report the crystal structure of human JNK1 complexed with pepJIP1, the peptide fragment of JIP1, revealing its selectivity for JNK1 over other MAPKs and the allosteric inhibition mechanism. The van der Waals contacts by the three residues (Pro157, Leu160, and Leu162) of pepJIP1 and the hydrogen bonding between Glu329 of JNK1 and Arg156 of pepJIP1 are critical for the selective binding. Binding of the peptide also induces a hinge motion between the N‐ and C‐terminal domains of JNK1 and distorts the ATP‐binding cleft, reducing the affinity of the kinase for ATP. In addition, we also determined the ternary complex structure of pepJIP1‐bound JNK1 complexed with SP600125, an ATP‐competitive inhibitor of JNK, providing the basis for the JNK specificity of the compound.

Crystal structure of the human centromeric nucleosome containing CENP-A

In eukaryotes, accurate chromosome segregation during mitosis and meiosis is coordinated by kinetochores, which are unique chromosomal sites for microtubule attachment. Centromeres specify the kinetochore formation sites on individual chromosomes, and are epigenetically marked by the assembly of nucleosomes containing the centromere-specific histone H3 variant, CENP-A. Although the underlying mechanism is unclear, centromere inheritance is probably dictated by the architecture of the centromeric nucleosome. Here we report the crystal structure of the human centromeric nucleosome containing CENP-A and its cognate α-satellite DNA derivative (147 base pairs). In the human CENP-A nucleosome, the DNA is wrapped around the histone octamer, consisting of two each of histones H2A, H2B, H4 and CENP-A, in a left-handed orientation. However, unlike the canonical H3 nucleosome, only the central 121 base pairs of the DNA are visible. The thirteen base pairs from both ends of the DNA are invisible in the crystal structure, and the αN helix of CENP-A is shorter than that of H3, which is known to be important for the orientation of the DNA ends in the canonical H3 nucleosome. A structural comparison of the CENP-A and H3 nucleosomes revealed that CENP-A contains two extra amino acid residues (Arg 80 and Gly 81) in the loop 1 region, which is completely exposed to the solvent. Mutations of the CENP-A loop 1 residues reduced CENP-A retention at the centromeres in human cells. Therefore, the CENP-A loop 1 may function in stabilizing the centromeric chromatin containing CENP-A, possibly by providing a binding site for trans-acting factors. The structure provides the first atomic-resolution picture of the centromere-specific nucleosome.

Structure of the catalytic domain of human phosphodiesterase 5 with bound drug molecules

Phosphodiesterases (PDEs) are a superfamily of enzymes that degrade the intracellular second messengers cyclic AMP and cyclic GMP. As essential regulators of cyclic nucleotide signalling with diverse physiological functions, PDEs are drug targets for the treatment of various diseases, including heart failure, depression, asthma, inflammation and erectile dysfunction. Of the 12 PDE gene families, cGMP-specific PDE5 carries out the principal cGMP-hydrolysing activity in human corpus cavernosum tissue. It is well known as the target of sildenafil citrate (Viagra) and other similar drugs for the treatment of erectile dysfunction. Despite the pressing need to develop selective PDE inhibitors as therapeutic drugs, only the cAMP-specific PDE4 structures are currently available. Here we present the three-dimensional structures of the catalytic domain (residues 537–860) of human PDE5 complexed with the three drug molecules sildenafil, tadalafil (Cialis) and vardenafil (Levitra). These structures will provide opportunities to design potent and selective PDE inhibitors with improved pharmacological profiles.

Crystal structure of hemoglobin protease, a heme binding autotransporter protein from pathogenic Escherichia coli

The acquisition of iron is essential for the survival of pathogenic bacteria, which have consequently evolved a wide variety of uptake systems to extract iron and heme from host proteins such as hemoglobin. Hemoglobin protease (Hbp) was discovered as a factor involved in the symbiosis of pathogenic Escherichia coli and Bacteroides fragilis, which cause intra-abdominal abscesses. Released from E. coli, this serine protease autotransporter degrades hemoglobin and delivers heme to both bacterial species. The crystal structure of the complete passenger domain of Hbp (110 kDa) is presented, which is the first structure from this class of serine proteases and the largest parallel β-helical structure yet solved.

Structural insight into the essential PB1-PB2 subunit contact of the influenza virus RNA polymerase

Influenza virus RNA‐dependent RNA polymerase is a multi‐functional heterotrimer, which uses a ‘cap‐snatching’ mechanism to produce viral mRNA. Host cell mRNA is cleaved to yield a cap‐bearing oligonucleotide, which can be extended using viral genomic RNA as a template. The cap‐binding and endonuclease activities are only activated once viral genomic RNA is bound. This requires signalling from the RNA‐binding PB1 subunit to the cap‐binding PB2 subunit, and the interface between these two subunits is essential for the polymerase activity. We have defined this interaction surface by protein crystallography and tested the effects of mutating contact residues on the function of the holo‐enzyme. This novel interface is surprisingly small, yet, it has a crucial function in regulating the 250 kDa polymerase complex and is completely conserved among avian and human influenza viruses.

Structural studies of a bacterial condensin complex reveal ATP-dependent disruption of intersubunit interactions.

Condensins are key mediators of chromosome condensation across organisms. Like other condensins, the bacterial MukBEF condensin complex consists of an SMC family protein dimer containing two ATPase head domains, MukB, and two interacting subunits, MukE and MukF. We report complete structural views of the intersubunit interactions of this condensin along with ensuing studies that reveal a role for the ATPase activity of MukB. MukE and MukF together form an elongated dimeric frame, and MukFs C-terminal winged-helix domains (C-WHDs) bind MukB heads to constitute closed ring-like structures. Surprisingly, one of the two bound C-WHDs is forced to detach upon ATP-mediated engagement of MukB heads. This detachment reaction depends on the linker segment preceding the C-WHD, and mutations on the linker restrict cell growth. Thus ATP-dependent transient disruption of the MukB-MukF interaction, which creates openings in condensin ring structures, is likely to be a critical feature of the functional mechanism of condensins.

Structural Mechanism for Inactivation and Activation of CAD/DFF40 in the Apoptotic Pathway

CAD/DFF40 is responsible for the degradation of chromosomal DNA into nucleosomal fragments and subsequent chromatin condensation during apoptosis. It exists as an inactive complex with its inhibitor ICAD/DFF45 in proliferating cells but becomes activated upon cleavage of ICAD/DFF45 into three domains by caspases in dying cells. The molecular mechanism underlying the control and activation of CAD/DFF40 was unknown. Here, the crystal structure of activated CAD/DFF40 reveals that it is a pair of molecular scissors with a deep active-site crevice that appears ideal for distinguishing internucleosomal DNA from nucleosomal DNA. Ensuing studies show that ICAD/DFF45 sequesters the nonfunctional CAD/DFF40 monomer and is also able to disassemble the functional CAD/DFF40 dimer. This capacity requires the involvement of the middle domain of ICAD/DFF45, which by itself cannot remain bound to CAD/DFF40 due to low binding affinity for the enzyme. Thus, the consequence of the caspase-cleavage of ICAD/DFF45 is a self-assembly of CAD/DFF40 into the active dimer.

Crystal structure of a clip‐domain serine protease and functional roles of the clip domains

Clip‐domain serine proteases (SPs) are the essential components of extracellular signaling cascades in various biological processes, especially in embryonic development and the innate immune responses of invertebrates. They consist of a chymotrypsin‐like SP domain and one or two clip domains at the N‐terminus. Prophenoloxidase‐activating factor (PPAF)‐II, which belongs to the noncatalytic clip‐domain SP family, is indispensable for the generation of the active phenoloxidase leading to melanization, a major defense mechanism of insects. Here, the crystal structure of PPAF‐II reveals that the clip domain adopts a novel fold containing a central cleft, which is distinct from the structures of defensins with a similar arrangement of cysteine residues. Ensuing studies demonstrated that PPAF‐II forms a homo‐oligomer upon cleavage by the upstream protease and that the clip domain of PPAF‐II functions as a module for binding phenoloxidase through the central cleft, while the clip domain of a catalytically active easter‐type SP plays an essential role in the rapid activation of its protease domain.

A Common Mechanism for the ATP-DnaA-dependent Formation of Open Complexes at the Replication Origin

Initiation of chromosomal replication and its cell cycle-coordinated regulation bear crucial and fundamental mechanisms in most cellular organisms. Escherichia coli DnaA protein forms a homomultimeric complex with the replication origin (oriC). ATP-DnaA multimers unwind the duplex within the oriC unwinding element (DUE). In this study, structural analyses suggested that several residues exposed in the central pore of the putative structure of DnaA multimers could be important for unwinding. Using mutation analyses, we found that, of these candidate residues, DnaA Val-211 and Arg-245 are prerequisites for initiation in vivo and in vitro. Whereas DnaA V211A and R245A proteins retained normal affinities for ATP/ADP and DNA and activity for the ATP-specific conformational change of the initiation complex in vitro, oriC complexes of these mutant proteins were inactive in DUE unwinding and in binding to the single-stranded DUE. Unlike oriC complexes including ADP-DnaA or the mutant DnaA, ATP-DnaA-oriC complexes specifically bound the upper strand of single-stranded DUE. Specific T-rich sequences within the strand were required for binding. The corresponding conserved residues of the DnaA ortholog in Thermotoga maritima, an ancient eubacterium, were also required for DUE unwinding, consistent with the idea that the mechanism and regulation for DUE unwinding can be evolutionarily conserved. These findings provide novel insights into mechanisms for pore-mediated origin unwinding, ATP/ADP-dependent regulation, and helicase loading of the initiation complex.

Visualizing breathing motion of internal cavities in concert with ligand migration in myoglobin

Proteins harbor a number of cavities of relatively small volume. Although these packing defects are associated with the thermodynamic instability of the proteins, the cavities also play specific roles in controlling protein functions, e.g., ligand migration and binding. This issue has been extensively studied in a well-known protein, myoglobin (Mb). Mb reversibly binds gas ligands at the heme site buried in the protein matrix and possesses several internal cavities in which ligand molecules can reside. It is still an open question as to how a ligand finds its migration pathways between the internal cavities. Here, we report on the dynamic and sequential structural deformation of internal cavities during the ligand migration process in Mb. Our method, the continuous illumination of native carbonmonoxy Mb crystals with pulsed laser at cryogenic temperatures, has revealed that the migration of the CO molecule into each cavity induces structural changes of the amino acid residues around the cavity, which results in the expansion of the cavity with a breathing motion. The sequential motion of the ligand and the cavity suggests a self-opening mechanism of the ligand migration channel arising by induced fit, which is further supported by computational geometry analysis by the Delaunay tessellation method. This result suggests a crucial role of the breathing motion of internal cavities as a general mechanism of ligand migration in a protein matrix.