Structural basis of ATG3 recognition by the autophagic ubiquitin-like protein ATG12

The autophagic ubiquitin-like protein (ublp) autophagy-related (ATG)12 is a component of the ATG12∼ATG5–ATG16L1 E3 complex that promotes lipid conjugation of members of the LC3 ublp family. A role of ATG12 in the E3 complex is to recruit the E2 enzyme ATG3. Here we report the identification of the ATG12 binding sequence in the flexible region of human ATG3 and the crystal structure of the minimal E3 complexed with the identified binding fragment of ATG3. The structure shows that 13 residues of the ATG3 fragment form a short β-strand followed by an α-helix on a surface area that is exclusive to ATG12. Mutational analyses of ATG3 confirm that four residues whose side chains make contacts with ATG12 are important for E3 interaction as well as LC3 lipidation. Conservation of these four critical residues is high in metazoan organisms and plants but lower in fungi. A structural comparison reveals that the ATG3 binding surface on ATG12 contains a hydrophobic pocket corresponding to the binding pocket of LC3 that accommodates the leucine of the LC3-interacting region motif. These findings establish the mechanism of ATG3 recruitment by ATG12 in higher eukaryotes and place ATG12 among the members of signaling ublps that bind liner sequences.Autophagy is a bulk degradation/recycling process essential for quality control in eukaryotic cells (1, 2). During autophagy, portions of cytoplasm are sequestered into membrane-bound vesicles called autophagosomes and transported into lysosomes for degradation. Autophagosome formation is mediated by a concerted action of a number of conserved autophagy-related (ATG) proteins (2, 3). The two classes of ubiquitin-like proteins (ublps) among these are members of the LC3 family in mammals or Atg8 in yeast and the conserved ATG12, which share some sequence similarities (4). LC3 and ATG12 are activated by the same E1 enzyme, ATG7, and then transferred to different E2 enzymes, ATG3 and ATG10, respectively. ATG3 conjugates LC3 to a lipid molecule, phosphatidylethanolamine (PE), on autophagosomal membranes. PE-conjugated LC3 plays crucial roles in control of membrane dynamics during autophagosome formation as well as in recruitment of cargos, such as aberrant proteins and damaged organelles, through binding to receptor proteins carrying an LC3-interacting region (LIR) motif (5). On the other hand, ATG10 attaches ATG12 to a structural protein, ATG5, and the resulting conjugate ATG12∼ATG5 acts like an E3 factor by stimulating the transfer of LC3 from ATG3 to PE (6). ATG12∼ATG5 exists as a complex with the coiled-coil protein ATG16L1 that localizes on autophagosome precursor membranes, resulting in LC3 lipidation for autophagosome formation (7). We previously showed that E3 activity requires the native covalent linkage between ATG12 and ATG5 and a composite surface patch formed by the residues of both of these proteins, and that the interaction with the E2 ATG3 is mostly mediated by ATG12 (8). However, it remains unclear how E3 communicates with or recruits E2.Here we describe atomic details of the ATG3–ATG12 interaction based on our crystal structure of a human E2–E3 complex containing this interaction. The results from accompanying interaction analyses and cellular LC3 lipidation assays support the importance of this interaction in LC3 lipidation and autophagy. We also show that the ATG3–ATG12 and LIR–LC3 interactions share some structural features. This unexpected finding together with our experimental data suggests that ATG12 is a protein recruitment apparatus in autophagy.     

Structural insights into the interaction of IL-33 with its receptors

Interleukin (IL)-33 is an important member of the IL-1 family that has pleiotropic activities in innate and adaptive immune responses in host defense and disease. It signals through its ligand-binding primary receptor ST2 and IL-1 receptor accessory protein (IL-1RAcP), both of which are members of the IL-1 receptor family. To clarify the interaction of IL-33 with its receptors, we determined the crystal structure of IL-33 in complex with the ectodomain of ST2 at a resolution of 3.27 Å. Coupled with structure-based mutagenesis and binding assay, the structural results define the molecular mechanism by which ST2 specifically recognizes IL-33. Structural comparison with other ligand–receptor complexes in the IL-1 family indicates that surface-charge complementarity is critical in determining ligand-binding specificity of IL-1 primary receptors. Combined crystallography and small-angle X-ray–scattering studies reveal that ST2 possesses hinge flexibility between the D3 domain and D1D2 module, whereas IL-1RAcP exhibits a rigid conformation in the unbound state in solution. The molecular flexibility of ST2 provides structural insights into domain-level conformational change of IL-1 primary receptors upon ligand binding, and the rigidity of IL-1RAcP explains its inability to bind ligands directly. The solution architecture of IL-33–ST2–IL-1RAcP complex from small-angle X-ray–scattering analysis resembles IL-1β–IL-1RII–IL-1RAcP and IL-1β–IL-1RI–IL-1RAcP crystal structures. The collective results confer IL-33 structure–function relationships, supporting and extending a general model for ligand–receptor assembly and activation in the IL-1 family.Interleukin (IL)-33 has important roles in initiating a type 2 immune response during infectious, inflammatory, and allergic diseases (1–5). It was initially identified as a nuclear factor in endothelial cells and named NF-HEV (nuclear factor from high endothelial venules) (6, 7). In 2005, it was rediscovered as a new member of the IL-1 family and an extracellular ligand for the orphan IL-1 receptor family member ST2 (8). As an extracellular cytokine, IL-33 is involved in the polarization of Th2 cells and activation of mast cells, basophils, eosinophils, and natural killer cells (1–3). Recent studies also discovered that the type 2 innate lymphoid cells (ILC2s) are major target cells of IL-33 (9, 10). ILC2s express a high level of ST2 and secrete large amounts of Th2 cytokines, most notably IL-5 and IL-13, when stimulated with IL-33 (11–13). Activation of ILC2s is essential in the initiation of the type 2 immune response against helminth infection and during allergic diseases such as asthma (9, 10).IL-33 does not have a signal peptide and is synthesized with an N-terminal propeptide upstream of the IL-1–like cytokine domain. It is preferentially and constitutively expressed in the nuclei of structural and lining cells, particularly in epithelial and endothelial cells (14, 15). Tissue damage caused by pathogen invasion or allergen exposure may lead to the release of IL-33 into extracellular environment from necrotic cells, which functions as an endogenous danger signal or alarmin (14, 16). Full-length human IL-33 consists of 270 residues and is biologically active (17, 18). It is also a substrate of serine proteases released by inflammatory cells recruited to the site of injury (18, 19). The proteases elastase, cathespin G, and proteinase 3 cleave full-length IL-33 to release N-terminal–truncated mature forms containing the IL-1–like cytokine domain: IL-33_95–270, l-33_99–270, and IL-33_109–270 (18). These mature IL-33 forms process a 10-fold greater potency to activate ST2 than full-length IL-33 (18). Caspase-1 was also suggested to cleave IL-33 to generate an active IL-33_112–270 that is the commercially available mature IL-33 form (8). However, it was later demonstrated that this cleavage site does not exist and cleavage by caspases at other sites actually inactivates IL-33 (17, 20, 21).The signaling of IL-33 depends on its binding to the primary receptor ST2 and subsequent recruitment of accessory receptor IL-1RAcP (8, 22, 23). The ligand-binding–induced receptor heterodimerization results in the juxtaposition of the intracellular toll/interleukin-1 receptor (TIR) domains of both receptors, which is necessary and sufficient to activate NF-κB and MAPK pathways in the target cells (24). Previously, we determined the complex structure of IL-1β with its decoy receptor IL-1RII and accessory receptor IL-1RAcP (25). Based on this structure and other previous studies, we proposed a general structural model for the assembly and activation of IL-1 family of cytokines with their receptors (25). In this model, ligand recognition relies on interaction of IL-1 cytokine with its primary receptor: IL-1α and IL-1β with IL-1RI; IL-33 with ST2; IL-18 with IL-18Rα; and IL-36α, IL-36β, and IL-36γ with IL-1Rrp2 (26–28). The binding forms a composite surface to recruit accessory receptor IL-1RAcP shared by IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and IL-36γ and IL-18Rβ by IL-18 (26, 27). This general structural model is further supported by the subsequent structural determination of IL-1β with IL-1RI and IL-1RAcP (29). However, there are still many key missing parts in the general structural model of ligand–receptor interaction in the IL-1 family. For example, the structural basis for specific recognition of IL-33 by ST2 and IL-18 by IL-18Rα, and promiscuous recognition of IL-36α, IL-36β, and IL-36γ by IL-1Rrp2 remains elusive. The proposed general model also needs further confirmation from structural studies of other signaling complexes in the IL-1 family. To address these issues, we studied the interaction of IL-33 with its receptors by a combination of X-ray crystallography and small-angle X-ray–scattering (SAXS) methods.     

Structural basis for the specific recognition of dual receptors by the homopolymeric pH 6 antigen (Psa) fimbriae of Yersinia pestis

The pH 6 antigen (Psa) of Yersinia pestis consists of fimbriae that bind to two receptors: β1-linked galactosyl residues in glycosphingolipids and the phosphocholine group in phospholipids. Despite the ubiquitous presence of either moiety on the surface of many mammalian cells, Y. pestis appears to prefer interacting with certain types of human cells, such as macrophages and alveolar epithelial cells of the lung. The molecular mechanism of this apparent selectivity is not clear. Site-directed mutagenesis of the consensus choline-binding motif in the sequence of PsaA, the subunit of the Psa fimbrial homopolymer, identified residues that abolish galactosylceramide binding, phosphatidylcholine binding, or both. The crystal structure of PsaA in complex with both galactose and phosphocholine reveals separate receptor binding sites that share a common structural motif, thus suggesting a potential interaction between the two sites. Mutagenesis of this shared structural motif identified Tyr126, which is part of the choline-binding consensus sequence but is found in direct contact with the galactose in the structure of PsaA, important for both receptor binding. Thus, this structure depicts a fimbrial subunit that forms a polymeric adhesin with a unique arrangement of dual receptor binding sites. These findings move the field forward by providing insights into unique types of multiple receptor–ligand interactions and should steer research into the synthesis of dual receptor inhibitor molecules to slow down the rapid progression of plague.     

Conformational Changes in Ff Phage Protein gVp upon Complexation with Its Viral Single-Stranded DNA Revealed Using Magic-Angle Spinning Solid-State NMR

Gene V protein (gVp) of the bacteriophages of the Ff family is a non-specific single-stranded DNA (ssDNA) binding protein. gVp binds to viral DNA during phage replication inside host Escherichia coli cells, thereby blocking further replication and signaling the assembly of new phage particles. gVp is a dimer in solution and in crystal form. A structural model of the complex between gVp and ssDNA was obtained via docking the free gVp to structures of short ssDNA segments and via the detection of residues involved in DNA binding in solution. Using solid-state NMR, we characterized structural features of the gVp in complex with full-length viral ssDNA. We show that gVp binds ssDNA with an average distance of 5.5 Å between the amino acid residues of the protein and the phosphate backbone of the DNA. Torsion angle predictions and chemical shift perturbations indicate that there were considerable structural changes throughout the protein upon complexation with ssDNA, with the most significant variations occurring at the ssDNA binding loop and the C-terminus. Our data suggests that the structure of gVp in complex with ssDNA differs significantly from the structure of gVp in the free form, presumably to allow for cooperative binding of dimers to form the filamentous phage particle.     

Substrate binding site flexibility of the small heat shock protein molecular chaperones

Small heat shock proteins (sHSPs) serve as a first line of defense against stress-induced cell damage by binding and maintaining denaturing proteins in a folding-competent state. In contrast to the well-defined substrate binding regions of ATP-dependent chaperones, interactions between sHSPs and substrates are poorly understood. Defining substrate-binding sites of sHSPs is key to understanding their cellular functions and to harnessing their aggregation-prevention properties for controlling damage due to stress and disease. We incorporated a photoactivatable cross-linker at 32 positions throughout a well-characterized sHSP, dodecameric PsHsp18.1 from pea, and identified direct interaction sites between sHSPs and substrates. Model substrates firefly luciferase and malate dehydrogenase form strong contacts with multiple residues in the sHSP N-terminal arm, demonstrating the importance of this flexible and evolutionary variable region in substrate binding. Within the conserved α-crystallin domain both substrates also bind the β-strand (β7) where mutations in human homologs result in inherited disease. Notably, these binding sites are poorly accessible in the sHSP atomic structure, consistent with major structural rearrangements being required for substrate binding. Detectable differences in the pattern of cross-linking intensity of the two substrates and the fact that substrates make contacts throughout the sHSP indicate that there is not a discrete substrate binding surface. Our results support a model in which the intrinsically-disordered N-terminal arm can present diverse geometries of interaction sites, which is likely critical for the ability of sHSPs to protect efficiently many different substrates.     

Structural basis for DNA recognition and loading into a viral packaging motor

Genome packaging into preformed viral procapsids is driven by powerful molecular motors. The small terminase protein is essential for the initial recognition of viral DNA and regulates the motor's ATPase and nuclease activities during DNA translocation. The crystal structure of a full-length small terminase protein from the Siphoviridae bacteriophage SF6, comprising the N-terminal DNA binding, the oligomerization core, and the C-terminal β-barrel domains, reveals a nine-subunit circular assembly in which the DNA-binding domains are arranged around the oligomerization core in a highly flexible manner. Mass spectrometry analysis and four further crystal structures show that, although the full-length protein exclusively forms nine-subunit assemblies, protein constructs missing the C-terminal β-barrel form both nine-subunit and ten-subunit assemblies, indicating the importance of the C terminus for defining the oligomeric state. The mechanism by which a ring-shaped small terminase oligomer binds viral DNA has not previously been elucidated. Here, we probed binding in vitro by using EPR and surface plasmon resonance experiments, which indicated that interaction with DNA is mediated exclusively by the DNA-binding domains and suggested a nucleosome-like model in which DNA binds around the outside of the protein oligomer.     

Molecular basis for the action of the collagen-specific chaperone Hsp47/SERPINH1 and its structure-specific client recognition

Collagen is the most abundant protein in animals and is a major component of the extracellular matrix in tissues such as skin and bone. A distinctive structural feature of all collagen types is a unique triple-helical structure formed by tandem repeats of the consensus sequence Xaa-Yaa-Gly, in which Xaa and Yaa frequently are proline and hydroxyproline, respectively. Hsp47/SERPINH1 is a procollagen-specific molecular chaperone that, unlike other chaperones, specifically recognizes the folded conformation of its client. Reduced functional levels of Hsp47 were reported in severe recessive forms of osteogenesis imperfecta, and homozygous knockout is lethal in mice. Here we present crystal structures of Hsp47 in its free form and in complex with homotrimeric synthetic collagen model peptides, each comprising one Hsp47-binding site represented by an arginine at the Yaa-position of a Xaa-Yaa-Gly triplet. Two of these three binding sites in the triple helix are occupied by Hsp47 molecules, which bind in a head-to-head fashion, thus making extensive contacts with the leading and trailing strands of the collagen triple helix. The important arginine residue within the Xaa-Arg-Gly triplet is recognized by a conserved aspartic acid. The structures explain the stabilization of the triple helix as well as the inhibition of collagen-bundle formation by Hsp47. In addition, we propose a pH-dependent substrate release mechanism based on a cluster of histidine residues.     

Structure of the enzyme-acyl carrier protein (ACP) substrate gatekeeper complex required for biotin synthesis

Although the pimeloyl moiety was long known to be a biotin precursor, the mechanism of assembly of this C7 α,ω-dicarboxylic acid was only recently elucidated. In Escherichia coli, pimelate is made by bypassing the strict specificity of the fatty acid synthetic pathway. BioC methylates the free carboxyl of a malonyl thioester, which replaces the usual acetyl thioester primer. This atypical primer is transformed to pimeloyl-acyl carrier protein (ACP) methyl ester by two cycles of fatty acid synthesis. The question is, what stops this product from undergoing further elongation? Although BioH readily cleaves this product in vitro, the enzyme is nonspecific, which made assignment of its physiological substrate problematical, especially because another enzyme, BioF, could also perform this gatekeeping function. We report the 2.05-Å resolution cocrystal structure of a complex of BioH with pimeloyl-ACP methyl ester and use the structure to demonstrate that BioH is the gatekeeper and its physiological substrate is pimeloyl-ACP methyl ester.     

Platform for in situ real-time measurement of protein-induced conformational changes of DNA

A platform for in situ and real-time measurement of protein-induced conformational changes in dsDNA is presented. We combine electrical orientation of surface-bound dsDNA probes with an optical technique to measure the kinetics of DNA conformational changes. The sequence-specific Escherichia coli integration host factor is utilized to demonstrate protein-induced bending upon binding of integration host factor to dsDNA probes. The effects of probe surface density on binding/bending kinetics are investigated. The platform can accommodate individual spots of microarrayed dsDNA on individually controlled, lithographically designed electrodes, making it amenable for use as a high throughput assay.