Structural basis for assembly and disassembly of the IGF/IGFBP/ALS ternary complex.

Insulin-like growth factors (IGFs) have pleiotropic roles in embryonic and postnatal growth and differentiation. Most serum IGFs are bound in a ternary complex with IGF-binding protein 3 (IGFBP3) and acid-labile subunit (ALS), extending the serum half-life of IGFs and regulating their availability. Here, we report cryo-EM structure of the human IGF1/IGFBP3/ALS ternary complex, revealing the detailed architecture of a parachute-like ternary complex and crucial determinants for their sequential and specific assembly. In vitro biochemical studies show that proteolysis at the central linker domain of IGFBP3 induces release of its C-terminal domain rather than IGF1 release from the ternary complex, yielding an intermediate complex that enhances IGF1 bioavailability. Our results provide mechanistic insight into IGF/IGFBP3/ALS ternary complex assembly and its disassembly upon proteolysis for IGF bioavailability, suggesting a structural basis for human diseases associated with IGF1 and IGFALS gene mutations such as complete ALS deficiency (ACLSD) and IGF1 deficiency.

Structural and Biochemical Analysis of the Furan Aldehyde Reductase YugJ from Bacillus subtilis.

NAD(H)/NADP(H)-dependent aldehyde/alcohol oxidoreductase (AAOR) participates in a wide range of physiologically important cellular processes by reducing aldehydes or oxidizing alcohols. Among AAOR substrates, furan aldehyde is highly toxic to microorganisms. To counteract the toxic effect of furan aldehyde, some bacteria have evolved AAOR that converts furan aldehyde into a less toxic alcohol. Based on biochemical and structural analyses, we identified Bacillus subtilis YugJ as an atypical AAOR that reduces furan aldehyde. YugJ displayed high substrate specificity toward 5-hydroxymethylfurfural (HMF), a furan aldehyde, in an NADPH- and Ni2+-dependent manner. YugJ folds into a two-domain structure consisting of a Rossmann-like domain and an α-helical domain. YugJ interacts with NADP and Ni2+ using the interdomain cleft of YugJ. A comparative analysis of three YugJ structures indicated that NADP(H) binding plays a key role in modulating the interdomain dynamics of YugJ. Noticeably, a nitrate ion was found in proximity to the nicotinamide ring of NADP in the YugJ structure, and the HMF-reducing activity of YugJ was inhibited by nitrate, providing insights into the substrate-binding mode of YugJ. These findings contribute to the characterization of the YugJ-mediated furan aldehyde reduction mechanism and to the rational design of improved furan aldehyde reductases for the biofuel industry.

Structural insights into the clustering and activation of Tie2 receptor mediated by Tie2 agonistic antibody.

Angiopoietin (Angpt)-Tie receptor 2 (Tie2) plays key roles in vascular development and homeostasis as well as pathological vascular remodeling. Therefore, Tie2-agonistic antibody and engineered Angpt1 variants have been developed as potential therapeutics for ischemic and inflammatory vascular diseases. However, their underlying mechanisms for Tie2 clustering and activation remain elusive and the poor manufacturability and stability of Angpt1 variants limit their clinical application. Here, we develop a human Tie2-agonistic antibody (hTAAB), which targets the membrane proximal fibronectin type III domain of Tie2 distinct from the Angpt-binding site. Our Tie2/hTAAB complex structures reveal that hTAAB tethers the preformed Tie2 homodimers into polygonal assemblies through specific binding to Tie2 Fn3 domain. Notably, the polygonal Tie2 clustering induced by hTAAB is critical for Tie2 activation and are resistant to antagonism by Angpt2. Our results provide insight into the molecular mechanism of Tie2 clustering and activation mediated by hTAAB, and the structure-based humanization of hTAAB creates a potential clinical application.

Structural study of the flagellar junction protein FlgL from Legionella pneumophila.

Legionella pneumophila is a flagellated pathogenic bacterium that causes atypical pneumonia called Legionnaires' disease. The flagellum plays a key role in the pathogenesis of L. pneumophila in the host. The protein FlgL forms a junction between the flagellar hook and filament and has been reported to elicit the host humoral immune response. To provide structural insights into FlgL-mediated junction assembly and FlgL-based vaccine design, we performed structural and serological studies on L. pneumophila FlgL (lpFlgL). The crystal structure of a truncated lpFlgL protein that consists of the D1 and D2 domains was determined at 3.06 Å resolution. The D1 domain of lpFlgL adopts a primarily helical, rod-shaped structure, and the D2 domain folds into a ß-sandwich structure that is affixed to the upper region of the D1 domain. The D1 domain of lpFlgL exhibits structural similarity to the flagellar filament protein flagellin, allowing us to propose a structural model of the lpFlgL junction based on the polymeric structure of flagellin. Furthermore, the D1 domain of lpFlgL exhibited substantially higher protein stability than the D2 domain and was responsible for most of the antigenicity of lpFlgL, suggesting that the D1 domain of lpFlgL would be a suitable target for the development of an anti-L. pneumophila vaccine.

Crystal structure of FlgL and its implications for flagellar assembly.

Bacteria move toward attractants and away from repellants by rotating their flagellum. The bacterial flagellum assembles through the ordered organization of more than 30 different proteins. Among the diverse flagellar proteins, FlgL forms the junction between the hook and the filament in the flagellum together with FlgK and provides a structural base where flagellin, a filament-forming protein, is inserted for the initiation of filament elongation. However, the functional and structural information available for FlgL is highly limited. To provide structural insights into the cross-linkage between the FlgL junction and the flagellin filament, we determined the crystal structures of FlgL from gram-positive Bacillus cereus (bcFlgL) and gram-negative Xanthomonas campestris (xcFlgL). bcFlgL contains one domain (D1), whereas xcFlgL adopts a two-domain structure that consists of the D1 and D2 domains. The constant D1 domain of FlgL adopts a rod structure that is generated by four longitudinal segments. This four-segment structure is recapitulated in filament and junction proteins but not in hook and rod proteins, allowing us to propose a junction-filament assembly mechanism based on a quasi-homotypic interaction. The D2 domain of xcFlgL resembles that of another junction protein, FlgK, suggesting the structural and functional relatedness of FlgL and FlgK.

Tetrameric structure of the flagellar cap protein FliD from Serratia marcescens.

Bacterial motility is provided by the flagellum. FliD is located at the distal end of the flagellum and plays a key role in the insertion of each flagellin protein at the growing tip of the flagellar filament. Because FliD functions as an oligomer, the determination of the oligomeric state of FliD is critical to understanding the molecular mechanism of FliD-mediated flagellar growth. FliD has been shown to adopt a pentameric or a hexameric structure depending on the bacterial species. Here, we report another distinct oligomeric form of FliD based on structural and biochemical studies. The crystal structures of the D2 and D3 domains of Serratia marcescens FliD (smFliD) were determined in two crystal forms and together revealed that smFliD assembles into a tetrameric architecture that resembles a four-pointed star plate. smFliD tetramerization was also confirmed in solution by cross-linking experiments. Although smFliD oligomerizes in a head-to-tail orientation using a common primary binding interface between the D2 and D3' domains (the prime denotes the second subunit in the oligomer) similarly to other FliD orthologs, the smFliD tetramer diverges to present a unique secondary D2-D2' binding interface. Our structure-based comparative analysis of FliD suggests that bacteria have developed diverse species-specific oligomeric forms of FliD that range from tetramers to hexamers for flagellar growth.

Self-Oligomerizing Structure of the Flagellar Cap Protein FliD and Its Implication in Filament Assembly.

FliD is a self-oligomerizing structural protein that caps the growing end of the bacterial flagellar filament. FliD also plays a key role in the flagellar system by continuously adding a new flagellin protein to the tip of the filament. To structurally characterize FliD oligomerization and to provide a FliD-mediated flagellin polymerization mechanism, we have determined the crystal structures of FliD proteins from Escherichia coli and Salmonella enterica serovar Typhimurium (ecFliD and stFliD, respectively). ecFliD consists of three domains (D1, D2, and D3) and forms a hexamer plate of the D2 and D3 domains that resembles a six-pointed star with legs consisting of the D1 domain. In contrast, the D2 and D3 domains of stFliD assemble into a pentamer as a five-pointed star plate. Despite their distinct oligomeric states, ecFliD and stFliD engage a common molecular surface for oligomerization. FliD also features interdomain and intersubunit flexibility, suggesting that FliD reorganizes its domains and adjacent subunits depending on the FliD binding partner. The similarity of the FliD shape to flagellin and the structural dynamics of FliD led us to propose a FliD-catalyzed filament elongation mechanism. In this model, FliD occupies a position in place of a nascent flagellin until the flagellin reaches the growing end of the filament, and then, FliD moves aside to repeat the positional replacement.