Glucosidase II and N-glycan mannose content regulate the half-lives of monoglucosylated species in vivo

Glucosidase II (GII) sequentially removes the two innermost glucose residues from the glycan (Glc(3)Man(9)GlcNAc(2)) transferred to proteins. GII also participates in cycles involving the lectin/chaperones calnexin (CNX) and calreticulin (CRT) as it removes the single glucose unit added to folding intermediates and misfolded glycoproteins by the UDP-Glc:glycoprotein glucosyltransferase (UGGT). GII is a heterodimer in which the α subunit (GIIα) bears the active site, and the β subunit (GIIβ) modulates GIIα activity through its C-terminal mannose 6-phosphate receptor homologous (MRH) domain. Here we report that, as already described in cell-free assays, in live Schizosaccharomyces pombe cells a decrease in the number of mannoses in the glycan results in decreased GII activity. Contrary to previously reported cell-free experiments, however, no such effect was observed in vivo for UGGT. We propose that endoplasmic reticulum α-mannosidase-mediated N-glycan demannosylation of misfolded/slow-folding glycoproteins may favor their interaction with the lectin/chaperone CNX present in S. pombe by prolonging the half-lives of the monoglucosylated glycans (S. pombe lacks CRT). Moreover, we show that even N-glycans bearing five mannoses may interact in vivo with the GIIβ MRH domain and that the N-terminal GIIβ G2B domain is involved in the GIIα-GIIβ interaction. Finally, we report that protists that transfer glycans with low mannose content to proteins have nevertheless conserved the possibility of displaying relatively long-lived monoglucosylated glycans by expressing GIIβ MRH domains with a higher specificity for glycans with high mannose content.     

Thermodynamic Characterization of the Interaction between Prefoldin and Group II Chaperonin

Prefoldin (PFD) is a hexameric chaperone that captures a protein substrate and transfers it to a group II chaperonin (CPN) to complete protein folding. We have studied the interaction between PFD and CPN using those from a hyperthermophilic archaeon, Thermococcus strain KS-1 (T. KS-1). In this study, we determined the crystal structure of the T. KS-1 PFDβ2 subunit and characterized the interactions between T. KS-1 CPNs (CPNα and CPNβ) and T. KS-1 PFDs (PFDα1-β1 and PFDα2-β2). As predicted from its amino acid sequence, the PFDβ2 subunit conforms to a structure similar to those of the PFDβ1 subunit and the Pyrococcus horikoshii OT3 PFDβ subunit, with the exception of the tip of its coiled-coil domain, which is thought to be the CPN interaction site. The interactions between T. KS-1 CPNs and PFDs (CPNα and PFDα1-β1; CPNα and PFDα2-β2; CPNβ and PFDα1-β1; and CPNβ and PFDα2-β2) were analyzed using the Biacore T100 system at various temperatures ranging from 20 to 45 ºC. The affinities between PFDs and CPNs increased with an increase in temperature. The thermodynamic parameters calculated from association constants showed that the interaction between PFD and CPN is entropy driven. Among the four combinations of PFD-CPN interactions, the entropy difference in binding between CPNβ and PFDα2-β2 was the largest, and affinity significantly increased at higher temperatures. Considering that expression of PFDα2-β2 and CPNβ subunit is induced upon heat shock, our results suggest that PFDα1-β1 is a general PFD for T. KS-1 CPNs, whereas PFDα2-β2 is specific for CPNβ.     

Nuclear but not cytosolic phosphoinositide 3-kinase beta has an essential function in cell survival

Class I(A) phosphoinositide 3-kinases (PI3Ks) are heterodimeric enzymes composed of a p85 regulatory and a p110 catalytic subunit that induce the formation of 3-polyphosphoinositides, which mediate cell survival, division, and migration. There are two ubiquitous PI3K isoforms p110α and p110β that have nonredundant functions in embryonic development and cell division. However, whereas p110α concentrates in the cytoplasm, p110β localizes to the nucleus and modulates nuclear processes such as DNA replication and repair. At present, the structural features that determine p110β nuclear localization remain unknown. We describe here that association with the p85β regulatory subunit controls p110β nuclear localization. We identified a nuclear localization signal (NLS) in p110β C2 domain that mediates its nuclear entry, as well as a nuclear export sequence (NES) in p85β. Deletion of p110β induced apoptosis, and complementation with the cytoplasmic C2-NLS p110β mutant was unable to restore cell survival. These studies show that p110β NLS and p85β NES regulate p85β/p110β nuclear localization, supporting the idea that nuclear, but not cytoplasmic, p110β controls cell survival.     

The human IKKbeta subunit kinase domain displays CK2-like phosphorylation specificity

NF-κB activation in response to pro-inflammatory stimuli relies upon phosphorylation of IκBα at serines 32 and 36 by the β subunit of the IκB kinase complex (IKK). In this study, we build upon the observation that highly purified human IKKβ subunit preparations retain this specificity in vitro. We show that IKKβ constructs that lack their carboxy-terminus beginning at the leucine zipper motif fail to phosphorylate IκBα at Ser-32 and Ser-36. Rather, these constructs, which contain the entire IKKβ subunit kinase domain, phosphorylate serine and threonine residues contained within the IκBα carboxy-terminal PEST region. Furthermore, removal of the leucine zipper and helix-loop-helix regions converts IKKβ to monomer. We propose that the helix-loop-helix of the human IKKβ subunit is necessary for restricting substrate specificity toward Ser-32 and Ser-36 in IκBα and that in the absence of its carboxy-terminal protein structural motifs the human IKKβ subunit kinase domain exhibits a CK2-like phosphorylation specificity.     

Structures of Hsp90α and Hsp90β bound to a purine-scaffold inhibitor reveal an exploitable residue for drug selectivity

Hsp90α and Hsp90β are implicated in a number of cancers and neurodegenerative disorders but the lack of selective pharmacological probes confounds efforts to identify their individual roles. Here, we analyzed the binding of an Hsp90α-selective PU compound, PU-11-trans, to the two cytosolic paralogs. We determined the co-crystal structures of Hsp90α and Hsp90β bound to PU-11-trans, as well as the structure of the apo Hsp90β NTD. The two inhibitor-bound structures reveal that Ser52, a nonconserved residue in the ATP binding pocket in Hsp90α, provides additional stability to PU-11-trans through a water-mediated hydrogen-bonding network. Mutation of Ser52 to alanine, as found in Hsp90β, alters the dissociation constant of Hsp90α for PU-11-trans to match that of Hsp90β. Our results provide a structural explanation for the binding preference of PU inhibitors for Hsp90α and demonstrate that the single nonconserved residue in the ATP-binding pocket may be exploited for α/β selectivity.     

Two homologous domains of similar structure but different stability in the yeast linker histone, Hho1p

The Saccharomyces cerevisiae homologue of the linker histone H1, Hho1p, has two domains that are similar in sequence to the globular domain of H1 (and variants such as H5). It is an open question whether both domains are functional and whether they play similar structural roles. Preliminary structural studies showed that the two isolated domains, GI and GII, differ significantly in stability. In 10 mM sodium phosphate (pH 7), the GI domain, like the globular domains of H1 and H5, GH1 and GH5, was stably folded, whereas GII was largely unstructured. However, at high concentrations of large tetrahedral anions (phosphate, sulphate, perchlorate), which might mimic the charge-screening effects of DNA phosphate groups, GII was folded. In view of the potential significance of these observations in relation to the role of Hho1p, we have now determined the structures of its GI and GII domains by NMR spectroscopy under conditions in which GII (like GI) is folded. The backbone r.m.s.d. over the ordered residues is 0.43 A for GI and 0.97 A for GII. Both structures show the "winged-helix" fold typical of GH1 and GH5 and are very similar to each other, with an r.m.s.d. over the structured regions of 1.3 A, although there are distinct differences. The potential for GII to adopt a structure similar to that of GI when Hho1p is bound to chromatin in vivo suggests that both globular domains might be functional. Whether Hho1p performs a structural role by bridging two nucleosomes remains to be determined.     

The crystal structure of maleylacetate reductase from Rhizobium sp. strain MTP‐10005 provides insights into the reaction mechanism of enzymes in its original family

Maleylacetate reductase plays a crucial role in catabolism of resorcinol by catalyzing the NAD(P)H‐dependent reduction of maleylacetate, at a carbon–carbon double bond, to 3‐oxoadipate. The crystal structure of maleylacetate reductase from Rhizobium sp. strain MTP‐10005, GraC, has been elucidated by the X‐ray diffraction method at 1.5 Å resolution. GraC is a homodimer, and each subunit consists of two domains: an N‐terminal NADH‐binding domain adopting an α/β structure and a C‐terminal functional domain adopting an α‐helical structure. Such structural features show similarity to those of the two existing families of enzymes in dehydroquinate synthase‐like superfamily. However, GraC is distinct in dimer formation and activity expression mechanism from the families of enzymes. Two subunits in GraC have different structures from each other in the present crystal. One subunit has several ligands mimicking NADH and the substrate in the cleft and adopts a closed domain arrangement. In contrast, the other subunit does not contain any ligand causing structural changes and adopts an open domain arrangement. The structure of GraC reveals those of maleylacetate reductase both in the coenzyme, substrate‐binding state and in the ligand‐free state. The comparison of both subunit structures reveals a conformational change of the Tyr326 loop for interaction with His243 on ligand binding. Structures of related enzymes suggest that His243 is likely a catalytic residue of GraC. Mutational analyses of His243 and Tyr326 support the catalytic roles proposed from structural information. The crystal structure of GraC characterizes the maleylacetate reductase family as a third family in the dehydroquinate synthase‐like superfamily. Proteins 2016; 84:1029–1042. © 2016 Wiley Periodicals, Inc.     

Surfactant protein D (Sp-D) binds to membrane-proximal domain (D3) of signal regulatory protein α (SIRPα), a site distant from binding domain of CD47, while also binding to analogous region on signal regulatory protein β (SIRPβ)

Signal regulatory protein α (SIRPα), a highly glycosylated type-1 transmembrane protein, is composed of three immunoglobulin-like extracellular loops as well as a cytoplasmic tail containing three classical tyrosine-based inhibitory motifs. Previous reports indicate that SIRPα binds to humoral pattern recognition molecules in the collectin family, namely surfactant proteins D and A (Sp-D and Sp-A, respectively), which are heavily expressed in the lung and constitute one of the first lines of innate immune defense against pathogens. However, little is known about molecular details of the structural interaction of Sp-D with SIRPs. In the present work, we examined the molecular basis of Sp-D binding to SIRPα using domain-deleted mutant proteins. We report that Sp-D binds to the membrane-proximal Ig domain (D3) of SIRPα in a calcium- and carbohydrate-dependent manner. Mutation of predicted N-glycosylation sites on SIRPα indicates that Sp-D binding is dependent on interactions with specific N-glycosylated residues on the membrane-proximal D3 domain of SIRPα. Given the remarkable sequence similarity of SIRPα to SIRPβ and the lack of known ligands for the latter, we examined Sp-D binding to SIRPβ. Here, we report specific binding of Sp-D to the membrane-proximal D3 domain of SIRPβ. Further studies confirmed that Sp-D binds to SIRPα expressed on human neutrophils and differentiated neutrophil-like cells. Because the other known ligand of SIRPα, CD47, binds to the membrane-distal domain D1, these findings indicate that multiple, distinct, functional ligand binding sites are present on SIRPα that may afford differential regulation of receptor function.     

Engineering the structural stability and functional properties of the GI domain into the intrinsically unfolded GII domain of the yeast linker histone Hho1p

Yeast Hho1p contains two domains, GI and GII, that are homologous to the single globular domain of the linker histone H1 (GH1). We showed previously that the isolated GI and GII domains have different structural stabilities and functional properties. GI, like GH1 and the related GH5, is stably folded at low ionic strength (10 mM sodium phosphate) and gives strong protection of chromatosome-length DNA ( approximately 166 bp) during micrococcal nuclease digestion of chromatin. GII is intrinsically unfolded in 10 mM sodium phosphate and gives weak chromatosome protection, but in 250 mM sodium phosphate has a structure very similar to that of GI as determined by NMR spectroscopy. We now show that the loop between helices II and III in GII is the cause of both its instability and its inability to confer strong chromatosome protection. A mutant GII, containing the loop of GI, termed GII-L, is stable in 10 mM sodium phosphate and is as effective as GI in chromatosome protection. Two GII mutants with selected mutations within the original loop were also slightly more stable than GII. In GII, two of the four basic residues conserved at the second DNA binding site ("site II") on the globular domain of canonical linker histones, and in GI, are absent. Introduction of the two "missing" site II basic residues into GII or GII-L destabilised the protein and led to decreased chromatosome protection relative to the protein without the basic residues. In general, the ability to confer chromatosome protection in vitro is closely related to structural stability (the relative population of structured and unstructured states). We have determined the structure of GII-L by NMR spectroscopy. GII-L is very similar to GII folded in 250 mM sodium phosphate, with the exception of the substituted loop region, which, as in GI, contains a single helical turn.     

胃癌细胞中视黄酸受体抑制AP-1活性的不同方式

 研究胃癌细胞中视黄酸受体RARα和RARβ抑制活化蛋白 1(activatorprotein 1,AP 1)活性的不同方式及其与全反式视黄酸 (ATRA)作用的相关性 .瞬时转染RARβ表达载体到MKN 4 5细胞后 ,佛波脂 (TPA)诱导的AP 1活性受到明显抑制 ,且与RARβ浓度正相关 ,与ATRA存在与否无关 ;相反 ,RARα转染细胞后 ,对TPA诱导的AP 1活性的抑制不仅与RARα的浓度相关 ,而且依赖于AT RA .凝胶阻抑测定表明 ,TPA可以显著加强AP 1结合活性 ,当ATRA处理不表达RARβ和低表达RARα的MKN 4 5细胞后 ,AP 1结合活性不受影响 ;然而 ,表达RARα和RARβ的BGC 82 3细胞经AT RA处理后 ,TPA诱导的AP 1结合活性则受到抑制 .另外 ,分析与抗AP 1活性相关的RARβ功能区表明 ,DNA结合区的缺失导致RARβ抑制AP 1活性作用的丧失 ,而配体结合区对于RARβ抑制AP 1活性则是非必需的 .以上结果证实 ,有胃癌细胞中 ,RARβ可能是AP 1活性的抑制因子 ,RARα则可能是ATRA作用的靶向 .尽管它们的作用方式有所不同 ,但最终都可以通过抑制AP 1活性来抑制胃癌细胞生长     

Mechanism of allosteric propagation across a β‐sheet structure investigated by molecular dynamics simulations

The bacterial adhesin FimH consists of an allosterically regulated mannose‐binding lectin domain and a covalently linked inhibitory pilin domain. Under normal conditions, the two domains are bound to each other, and FimH interacts weakly with mannose. However, under tensile force, the domains separate and the lectin domain undergoes conformational changes that strengthen its bond with mannose. Comparison of the crystallographic structures of the low and the high affinity state of the lectin domain reveals conformational changes mainly in the regulatory inter‐domain region, the mannose binding site and a large β sheet that connects the two distally located regions. Here, molecular dynamics simulations investigated how conformational changes are propagated within and between different regions of the lectin domain. It was found that the inter‐domain region moves towards the high affinity conformation as it becomes more compact and buries exposed hydrophobic surface after separation of the pilin domain. The mannose binding site was more rigid in the high affinity state, which prevented water penetration into the pocket. The large central β sheet demonstrated a soft spring‐like twisting. Its twisting motion was moderately correlated to fluctuations in both the regulatory and the binding region, whereas a weak correlation was seen in a direct comparison of these two distal sites. The results suggest a so called “population shift” model whereby binding of the lectin domain to either the pilin domain or mannose locks the β sheet in a rather twisted or flat conformation, stabilizing the low or the high affinity state, respectively. Proteins 2016; 84:990–1008. © 2016 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.     

Novel Isoform-Specific Interfaces Revealed by PKA RIIβ Holoenzyme Structures

The cAMP-dependent protein kinase catalytic (C) subunit is inhibited by two classes of functionally nonredundant regulatory (R) subunits, RI and RII. Unlike RI subunits, RII subunits are both substrates and inhibitors. Because RIIβ knockout mice have important disease phenotypes, the RIIβ holoenzyme is a target for developing isoform-specific agonists and/or antagonists. We also know little about the linker region that connects the inhibitor site to the N-terminal dimerization domain, although this linker determines the unique globular architecture of the RIIβ holoenzyme. To understand how RIIβ functions as both an inhibitor and a substrate and to elucidate the structural role of the linker, we engineered different RIIβ constructs. In the absence of nucleotide, RIIβ(108-268), which contains a single cyclic nucleotide binding domain, bound C subunit poorly, whereas with AMP-PNP, a non-hydrolyzable ATP analog, the affinity was 11 nM. The RIIβ(108-268) holoenzyme structure (1.62 Å) with AMP-PNP/Mn²⁺ showed that we trapped the RIIβ subunit in an enzyme:substrate complex with the C subunit in a closed conformation. The enhanced affinity afforded by AMP-PNP/Mn²⁺ may be a useful strategy for increasing affinity and trapping other protein substrates with their cognate protein kinase. Because mutagenesis predicted that the region N-terminal to the inhibitor site might dock differently to RI and RII, we also engineered RIIβ(102-265), which contained six additional linker residues. The additional linker residues in RIIβ(102-265) increased the affinity to 1.6 nM, suggesting that docking to this surface may also enhance catalytic efficiency. In the corresponding holoenzyme structure, this linker docks as an extended strand onto the surface of the large lobe. This hydrophobic pocket, formed by the αF-αG loop and conserved in many protein kinases, also provides a docking site for the amphipathic helix of PKI. This novel orientation of the linker peptide provides the first clues as to how this region contributes to the unique organization of the RIIβ holoenzyme.     

Structure of full‐length class I chitinase from rice revealed by X‐ray crystallography and small‐angle X‐ray scattering

The rice class I chitinase OsChia1b, also referred to as RCC2 or Cht‐2, is composed of an N‐terminal chitin‐binding domain (ChBD) and a C‐terminal catalytic domain (CatD), which are connected by a proline‐ and threonine‐rich linker peptide. Because of the ability to inhibit fungal growth, the OsChia1b gene has been used to produce transgenic plants with enhanced disease resistance. As an initial step toward elucidating the mechanism of hydrolytic action and antifungal activity, the full‐length structure of OsChia1b was analyzed by X‐ray crystallography and small‐angle X‐ray scattering (SAXS). We determined the crystal structure of full‐length OsChia1b at 2.00‐Å resolution, but there are two possibilities for a biological molecule with and without interdomain contacts. The SAXS data showed an extended structure of OsChia1b in solution compared to that in the crystal form. This extension could be caused by the conformational flexibility of the linker. A docking simulation of ChBD with tri‐N‐acetylchitotriose exhibited a similar binding mode to the one observed in the crystal structure of a two‐domain plant lectin complexed with a chitooligosaccharide. A hypothetical model based on the binding mode suggested that ChBD is unsuitable for binding to crystalline α‐chitin, which is a major component of fungal cell walls because of its collisions with the chitin chains on the flat surface of α‐chitin. This model also indicates the difference in the binding specificity of plant and bacterial ChBDs of GH19 chitinases, which contribute to antifungal activity. Proteins 2010. © 2010 Wiley‐Liss,Inc.     

Structure of the ribosome associating GTPase HflX

The HflX‐family is a widely distributed but poorly characterized family of translation factor‐related guanosine triphosphatases (GTPases) that interact with the large ribosomal subunit. This study describes the crystal structure of HflX from Sulfolobus solfataricus solved to 2.0‐Å resolution in apo‐ and GDP‐bound forms. The enzyme displays a two‐domain architecture with a novel “HflX domain” at the N‐terminus, and a classical G‐domain at the C‐terminus. The HflX domain is composed of a four‐stranded parallel β‐sheet flanked by two α‐helices on either side, and an anti‐parallel coiled coil of two long α‐helices that lead to the G‐domain. The cleft between the two domains accommodates the nucleotide binding site as well as the switch II region, which mediates interactions between the two domains. Conformational changes of the switch regions are therefore anticipated to reposition the HflX‐domain upon GTP‐binding. Slow GTPase activity has been confirmed, with an HflX domain deletion mutant exhibiting a 24‐fold enhanced turnover rate, suggesting a regulatory role for the HflX domain. The conserved positively charged surface patches of the HflX‐domain may mediate interaction with the large ribosomal subunit. The present study provides a structural basis to uncover the functional role of this GTPases family whose function is largely unknown. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Phosphatidylinositol Transfer Protein, Cytoplasmic 1 (PITPNC1) Binds and Transfers Phosphatidic Acid

Phosphatidylinositol transfer proteins (PITPs) are versatile proteins required for signal transduction and membrane traffic. The best characterized mammalian PITPs are the Class I PITPs, PITPα (PITPNA) and PITPβ (PITPNB), which are single domain proteins with a hydrophobic cavity that binds a phosphatidylinositol (PI) or phosphatidylcholine molecule. In this study, we report the lipid binding properties of an uncharacterized soluble PITP, phosphatidylinositol transfer protein, cytoplasmic 1 (PITPNC1) (alternative name, RdgBβ), of the Class II family. We show that the lipid binding properties of this protein are distinct to Class I PITPs because, besides PI, RdgBβ binds and transfers phosphatidic acid (PA) but hardly binds phosphatidylcholine. RdgBβ when purified from Escherichia coli is preloaded with PA and phosphatidylglycerol. When RdgBβ was incubated with permeabilized HL60 cells, phosphatidylglycerol was released, and PA and PI were now incorporated into RdgBβ. After an increase in PA levels following activation of endogenous phospholipase D or after addition of bacterial phospholipase D, binding of PA to RdgBβ was greater at the expense of PI binding. We propose that RdgBβ, when containing PA, regulates an effector protein or can facilitate lipid transfer between membrane compartments.     

Idiosyncrasy and identity in the prokaryotic Phe-system: crystal structure of E. coli phenylalanyl-tRNA synthetase complexed with phenylalanine and AMP

The crystal structure of Phenylalanyl‐tRNA synthetase from E. coli (EcPheRS), a class II aminoacyl‐tRNA synthetase, complexed with phenylalanine and AMP was determined at 3.05 Å resolution. EcPheRS is a (αβ)₂ heterotetramer: the αβ heterodimer of EcPheRS consists of 11 structural domains. Three of them: the N‐terminus, A1 and A2 belong to the α‐subunit and B1‐B8 domains to the β subunit. The structure of EcPheRS revealed that architecture of four helix‐bundle interface, characteristic of class IIc heterotetrameric aaRSs, is changed: each of the two long helices belonging to CLM transformed into the coil‐short helix structural fragments. The N‐terminal domain of the α‐subunit in EcPheRS forms compact triple helix domain. This observation is contradictory to the structure of the apo form of TtPheRS, where N‐terminal domain was not detected in the electron density map. Comparison of EcPheRS structure with TtPheRS has uncovered significant rearrangements of the structural domains involved in tRNA^Phe binding/translocation. As it follows from modeling experiments, to achieve a tighter fit with anticodon loop of tRNA, a shift of ～5 Å is required for C‐terminal domain B8, and of ～6 to 7 Å for the whole N terminus. EcPheRSs have emerged as an important target for the incorporation of novel amino acids into genetic code. Further progress in design of novel compounds is anticipated based on the structural data of EcPheRS.