Young Jun Im

Structural mechanism for sterol sensing and transport by OSBP-related proteins

The oxysterol-binding-protein (OSBP)-related proteins (ORPs) are conserved from yeast to humans, and are implicated in the regulation of sterol homeostasis and in signal transduction pathways. Here we report the structure of the full-length yeast ORP Osh4 (also known as Kes1) at 1.5–1.9 Å resolution in complexes with ergosterol, cholesterol, and 7-, 20- and 25-hydroxycholesterol. We find that a single sterol molecule binds within a hydrophobic tunnel in a manner consistent with a transport function for ORPs. The entrance is blocked by a flexible amino-terminal lid and surrounded by basic residues that are critical for Osh4 function. The structure of the open state of a lid-truncated form of Osh4 was determined at 2.5 Å resolution. Structural analysis and limited proteolysis show that sterol binding closes the lid and stabilizes a conformation favouring transport across aqueous barriers and signal transmission. The structure of Osh4 in the absence of ligand exposes potential phospholipid-binding sites that are positioned for membrane docking and sterol exchange. On the basis of these observations, we propose a model in which sterol and membrane binding promote reciprocal conformational changes that facilitate a sterol transfer and signalling cycle.

Crystal structures of the HslVU peptidase-ATPase complex reveal an ATP-dependent proteolysis mechanism.

BACKGROUND The bacterial heat shock locus HslU ATPase and HslV peptidase together form an ATP-dependent HslVU protease. Bacterial HslVU is a homolog of the eukaryotic 26S proteasome. Crystallographic studies of HslVU should provide an understanding of ATP-dependent protein unfolding, translocation, and proteolysis by this and other ATP-dependent proteases. RESULTS We present a 3.0 A resolution crystal structure of HslVU with an HslU hexamer bound at one end of an HslV dodecamer. The structure shows that the central pores of the ATPase and peptidase are next to each other and aligned. The central pore of HslU consists of a GYVG motif, which is conserved among protease-associated ATPases. The binding of one HslU hexamer to one end of an HslV dodecamer in the 3.0 A resolution structure opens both HslV central pores and induces asymmetric changes in HslV. CONCLUSIONS Analysis of nucleotide binding induced conformational changes in the current and previous HslU structures suggests a protein unfolding-coupled translocation mechanism. In this mechanism, unfolded polypeptides are threaded through the aligned pores of the ATPase and peptidase and translocated into the peptidase central chamber.

Nonvesicular sterol movement from plasma membrane to ER requires oxysterol-binding protein–related proteins and phosphoinositides

Sterols are moved between cellular membranes by nonvesicular pathways whose functions are poorly understood. In yeast, one such pathway transfers sterols from the plasma membrane (PM) to the endoplasmic reticulum (ER). We show that this transport requires oxysterol-binding protein (OSBP)–related proteins (ORPs), which are a large family of conserved lipid-binding proteins. We demonstrate that a representative member of this family, Osh4p/Kes1p, specifically facilitates the nonvesicular transfer of cholesterol and ergosterol between membranes in vitro. In addition, Osh4p transfers sterols more rapidly between membranes containing phosphoinositides (PIPs), suggesting that PIPs regulate sterol transport by ORPs. We confirmed this by showing that PM to ER sterol transport slows dramatically in mutants with conditional defects in PIP biosynthesis. Our findings argue that ORPs move sterols among cellular compartments and that sterol transport and intracellular distribution are regulated by PIPs.

Crystal structure of the Shank PDZ-ligand complex reveals a class I PDZ interaction and a novel PDZ-PDZ dimerization

The Shank/proline-rich synapse-associated protein family of multidomain proteins is known to play an important role in the organization of synaptic multiprotein complexes. For instance, the Shank PDZ domain binds to the C termini of guanylate kinase-associated proteins, which in turn interact with the guanylate kinase domain of postsynaptic density-95 scaffolding proteins. Here we describe the crystal structures of Shank1 PDZ in its peptide free form and in complex with the C-terminal hexapeptide (EAQTRL) of guanylate kinase-associated protein (GKAP1a) determined at 1.8- and 2.25-Å resolutions, respectively. The structure shows the typical class I PDZ interaction of PDZ-peptide complex with the consensus sequence -X-(Thr/Ser)-X-Leu. In addition, Asp-634 within the Shank1 PDZ domain recognizes the positively charged Arg at –1 position and hydrogen bonds, and salt bridges between Arg-607 and the side chains of the ligand at –3 and –5 positions contribute further to the recognition of the peptide ligand. Remarkably, whether free or complexed, Shank1 PDZ domains form dimers with a conserved βB/βC loop and N-terminal βA strands, suggesting a novel model of PDZ-PDZ homodimerization. This implies that antiparallel dimerization through the N-terminal βA strands could be a common configuration among PDZ dimers. Within the dimeric structure, the two-peptide binding sites are arranged so that the N termini of the bound peptide ligands are in close proximity and oriented toward the 2-fold axis of the dimer. This configuration may provide a means of facilitating dimeric organization of PDZ-target assemblies.

Structure and function of the ESCRT-II-III interface in multivesicular body biogenesis.

The ESCRT-II-ESCRT-III interaction coordinates the sorting of ubiquitinated cargo with the budding and scission of intralumenal vesicles into multivesicular bodies. The interacting regions of these complexes were mapped to the second winged helix domain of human ESCRT-II subunit VPS25 and the first helix of ESCRT-III subunit VPS20. The crystal structure of this complex was determined at 2.0 A resolution. Residues involved in structural interactions explain the specificity of ESCRT-II for Vps20, and are critical for cargo sorting in vivo. ESCRT-II directly activates ESCRT-III-driven vesicle budding and scission in vitro via these structural interactions. VPS20 and ESCRT-II bind membranes with nanomolar affinity, explaining why binding to ESCRT-II is dispensable for the recruitment of Vps20 to membranes. Docking of the ESCRT-II-VPS20(2) supercomplex reveals a convex membrane-binding surface, suggesting a hypothesis for negative membrane curvature induction in the nascent intralumenal vesicle.

Integrated structural model and membrane targeting mechanism of the human ESCRT-II complex

ESCRT-II plays a pivotal role in receptor downregulation and multivesicular body biogenesis and is conserved from yeast to humans. The crystal structures of two human ESCRT-II complex structures have been determined at 2.6 and 2.9 A resolution, respectively. The complex has three lobes and contains one copy each of VPS22 and VPS36 and two copies of VPS25. The structure reveals a dynamic helical domain to which both the VPS22 and VPS36 subunits contribute that connects the GLUE domain to the rest of the ESCRT-II core. Hydrodynamic analysis shows that intact ESCRT-II has a compact, closed conformation. ESCRT-II binds to the ESCRT-I VPS28 C-terminal domain subunit through a helix immediately C-terminal to the VPS36-GLUE domain. ESCRT-II is targeted to endosomal membranes by the lipid-binding activities of both the Vps36 GLUE domain and the first helix of Vps22. These data provide a unifying structural and functional framework for the ESCRT-II complex.

The Active Site of a Lon Protease from Methanococcus jannaschii Distinctly Differs from the Canonical Catalytic Dyad of Lon Proteases

ATP-dependent Lon proteases catalyze the degradation of various regulatory proteins and abnormal proteins within cells. Methanococcus jannaschii Lon (Mj-Lon) is a homologue of Escherichia coli Lon (Ec-Lon) but has two transmembrane helices within its N-terminal ATPase domain. We solved the crystal structure of the proteolytic domain of Mj-Lon using multiwavelength anomalous dispersion, refining it to 1.9-Å resolution. The structure displays an overall fold conserved in the proteolytic domain of Ec-Lon; however, the active site shows uniquely configured catalytic Ser-Lys-Asp residues that are not seen in Ec-Lon, which contains a catalytic dyad. In Mj-Lon, the C-terminal half of the β4-α2 segment is an α-helix, whereas it is a β-strand in Ec-Lon. Consequently, the configurations of the active sites differ due to the formation of a salt bridge between Asp-547 and Lys-593 in Mj-Lon. Moreover, unlike Ec-Lon, Mj-Lon has a buried cavity in the region of the active site containing three water molecules, one of which is hydrogen-bonded to catalytic Ser-550. The geometry and environment of the active site residues in Mj-Lon suggest that the charged Lys-593 assists in lowering the pKa of the Ser-550 hydroxyl group via its electrostatic potential, and the water in the cavity acts as a proton acceptor during catalysis. Extensive sequence alignment and comparison of the structures of the proteolytic domains clearly indicate that Lon proteases can be classified into two groups depending on active site configuration and the presence of DGPSA or (D/E)GDSA consensus sequences, as represented by Ec-Lon and Mj-Lon.

Structural mechanism of ergosterol regulation by fungal sterol transcription factor Upc2

Transcriptional regulation of ergosterol biosynthesis in fungi is crucial for sterol homeostasis and for resistance to azole drugs. In Saccharomyces cerevisiae, the Upc2 transcription factor activates the expression of related genes in response to sterol depletion by poorly understood mechanisms. We have determined the structure of the C-terminal domain (CTD) of Upc2, which displays a novel α-helical fold with a deep hydrophobic pocket. We discovered that the conserved CTD is a ligand-binding domain and senses the ergosterol level in the cell. Ergosterol binding represses its transcription activity, while dissociation of the ligand leads to relocalization of Upc2 from cytosol to nucleus for transcriptional activation. The C-terminal activation loop is essential for ligand binding and for transcriptional regulation. Our findings highlight that Upc2 represents a novel class of fungal zinc cluster transcription factors, which can serve as a target for the developments of antifungal therapeutics.

Crystal structure of a cyanobacterial phytochrome response regulator

The two‐component signal transduction pathway widespread in prokaryotes, fungi, molds, and some plants involves an elaborate phosphorelay cascade. Rcp1 is the phosphate receiver module in a two‐component system controlling the light response of cyanobacteria Synechocystis sp. via cyanobacterial phytochrome Cph1, which recognizes Rcp1 and transfers its phosphoryl group to an aspartate residue in response to light. Here we describe the crystal structure of Rcp1 refined to a crystallographic R‐factor of 18.8% at a resolution of 1.9 Å. The structure reveals a tightly associated homodimer with monomers comprised of doubly wound five‐stranded parallel β‐sheets forming a single‐domain protein homologous with the N‐terminal activator domain of other response regulators (e.g., chemotaxis protein CheY). The three‐dimensional structure of Rcp1 appears consistent with the conserved activation mechanism of phosphate receiver proteins, although in this case, the C‐terminal half of its regulatory domain, which undergoes structural changes upon phosphorylation, contributes to the dimerization interface. The involvement of the residues undergoing phosphorylation‐induced conformational changes at the dimeric interface suggests that dimerization of Rcp1 may be regulated by phosphorylation, which could affect the interaction of Rcp1 with downstream target molecules.

Structural Basis for Asymmetric Association of the βPIX Coiled Coil and Shank PDZ

betaPIX (p21-activated kinase interacting exchange factor) and Shank/ProSAP protein form a complex acting as a protein scaffold that integrates signaling pathways and regulates postsynaptic structure. Complex formation is mediated by the C-terminal PDZ binding motif of betaPIX and the Shank PDZ domain. The coiled-coil (CC) domain upstream of the PDZ binding motif allows multimerization of betaPIX, which is important for its physiological functions. We have solved the crystal structure of the betaPIX CC-Shank PDZ complex and determined the stoichiometry of complex formation. The betaPIX CC forms a 76-A-long parallel CC trimer. Despite the fact that the betaPIX CC exposes three PDZ binding motifs in the C-termini, the betaPIX trimer associates with a single Shank PDZ. One of the C-terminal ends of the CC forms an extensive beta-sheet interaction with the Shank PDZ, while the other two ends are not involved in ligand binding and form random coils. The two C-terminal ends of betaPIX have significantly lower affinity than the first PDZ binding motif due to the steric hindrance in the C-terminal tails, which results in binding of a single PDZ domain to the betaPIX trimer. The structure shows canonical class I PDZ binding with a beta-sheet interaction extending to position -6 of betaPIX. The betaB-betaC loop of Shank PDZ undergoes a conformational change upon ligand binding to form the beta-sheet interaction and to accommodate the bulky side chain of Trp -5. This structural study provides a clear picture of the molecular recognition of the PDZ ligand and the asymmetric association of betaPIX CC and Shank PDZ.