Histidyl-tRNA synthetase.

Histidyl-tRNA synthetase (HisRS) is responsible for the synthesis of histidyl-transfer RNA, which is essential for the incorporation of histidine into proteins. This amino acid has uniquely moderate basic properties and is an important group in many catalytic functions of enzymes. A compilation of currently known primary structures of HisRS shows that the subunits of these homo-dimeric enzymes consist of 420-550 amino acid residues. This represents a relatively short chain length among aminoacyl-tRNA synthetases (aaRS), whose peptide chain sizes range from about 300 to 1100 amino acid residues. The crystal structures of HisRS from two organisms and their complexes with histidine, histidyl-adenylate and histidinol with ATP have been solved. HisRS from Escherichia coli and Thermus thermophilus are very similar dimeric enzymes consisting of three domains: the N-terminal catalytic domain containing the six-stranded antiparallel beta-sheet and the three motifs characteristic of class II aaRS, a HisRS-specific helical domain inserted between motifs 2 and 3 that may contact the acceptor stem of the tRNA, and a C-terminal alpha/beta domain that may be involved in the recognition of the anticodon stem and loop of tRNA(His). The aminoacylation reaction follows the standard two-step mechanism. HisRS also belongs to the group of aaRS that can rapidly synthesize diadenosine tetraphosphate, a compound that is suspected to be involved in several regulatory mechanisms of cell metabolism. Many analogs of histidine have been tested for their properties as substrates or inhibitors of HisRS, leading to the elucidation of structure-activity relationships concerning configuration, importance of the carboxy and amino group, and the nature of the side chain. HisRS has been found to act as a particularly important antigen in autoimmune diseases such as rheumatic arthritis or myositis. Successful attempts have been made to identify epitopes responsible for the complexation with such auto-antibodies.     

Uncharged tRNA activates GCN2 by displacing the protein kinase moiety from a bipartite tRNA-binding domain

Protein kinase GCN2 regulates translation in amino acid-starved cells by phosphorylating elF2. GCN2 contains a regulatory domain related to histidyl-tRNA synthetase (HisRS) postulated to bind multiple deacylated tRNAs as a general sensor of starvation. In accordance with this model, GCN2 bound several deacylated tRNAs with similar affinities, and aminoacylation of tRNAphe weakened its interaction with GCN2. Unexpectedly, the C-terminal ribosome binding segment of GCN2 (C-term) was required in addition to the HisRS domain for strong tRNA binding. A combined HisRS+ C-term segment bound to the isolated protein kinase (PK) domain in vitro, and tRNA impeded this interaction. An activating mutation (GCN2c-E803V) that weakens PK-C-term association greatly enhanced tRNA binding by GCN2. These results provide strong evidence that tRNA stimulates the GCN2 kinase moiety by preventing an inhibitory interaction with the bipartite tRNA binding domain.     

Intraphylum diversity and complex evolution of cyanobacterial aminoacyl-tRNA synthetases

A comparative genomic analysis of 35 cyanobacterial strains has revealed that the gene complement of aminoacyl-tRNA synthetases (AARSs) and routes for aminoacyl-tRNA synthesis may differ among the species of this phylum. Several genes encoding AARS paralogues were identified in some genomes. In-depth phylogenetic analysis was done for each of these proteins to gain insight into their evolutionary history. GluRS, HisRS, ArgRS, ThrRS, CysRS, and Glu-Q-RS showed evidence of a complex evolutionary course as indicated by a number of inconsistencies with our reference tree for cyanobacterial phylogeny. In addition to sequence data, support for evolutionary hypotheses involving horizontal gene transfer or gene duplication events was obtained from other observations including biased sequence conservation, the presence of indels (insertions or deletions), or vestigial traces of ancestral redundant genes. We present evidences for a novel protein domain with two putative transmembrane helices recruited independently by distinct AARS in particular cyanobacteria.     

The tRNA-binding moiety in GCN2 contains a dimerization domain that interacts with the kinase domain and is required for tRNA binding and kinase activation

GCN2 stimulates translation of GCN4 mRNA in amino acid-starved cells by phosphorylating translation initiation factor 2. GCN2 is activated by binding of uncharged tRNA to a domain related to histidyl-tRNA synthetase (HisRS). The HisRS-like region contains two dimerization domains (HisRS-N and HisRS-C) required for GCN2 function in vivo but dispensable for dimerization by full-length GCN2. Residues corresponding to amino acids at the dimer interface of Escherichia coli HisRS were required for dimerization of recombinant HisRS-N and for tRNA binding by full-length GCN2, suggesting that HisRS-N dimerization promotes tRNA binding and kinase activation. HisRS-N also interacted with the protein kinase (PK) domain, and a deletion impairing this interaction destroyed GCN2 function without reducing tRNA binding; thus, HisRS-N-PK interaction appears to stimulate PK function. The C-terminal domain of GCN2 (C-term) interacted with the PK domain in a manner disrupted by an activating PK mutation (E803V). These results suggest that the C-term is an autoinhibitory domain, counteracted by tRNA binding. We conclude that multiple domain interactions, positive and negative, mediate the activation of GCN2 by uncharged tRNA.     

Interaction between the tRNA-Binding and C-Terminal Domains of Yeast Gcn2 Regulates Kinase Activity In Vivo

The stress-activated protein kinase Gcn2 regulates protein synthesis by phosphorylation of translation initiation factor eIF2α. Gcn2 is activated in amino acid-deprived cells by binding of uncharged tRNA to the regulatory domain related to histidyl-tRNA synthetase, but the molecular mechanism of activation is unclear. We used a genetic approach to identify a key regulatory surface in Gcn2 that is proximal to the predicted active site of the HisRS domain and likely remodeled by tRNA binding. Mutations leading to amino acid substitutions on this surface were identified that activate Gcn2 at low levels of tRNA binding (Gcd^- phenotype), while other substitutions block kinase activation (Gcn^- phenotype), in some cases without altering tRNA binding by Gcn2 in vitro. Remarkably, the Gcn^- substitutions increase affinity of the HisRS domain for the C-terminal domain (CTD), previously implicated as a kinase autoinhibitory segment, in a manner dampened by HisRS domain Gcd^- substitutions and by amino acid starvation in vivo. Moreover, tRNA specifically antagonizes HisRS/CTD association in vitro. These findings support a model wherein HisRS-CTD interaction facilitates the autoinhibitory function of the CTD in nonstarvation conditions, with tRNA binding eliciting kinase activation by weakening HisRS-CTD association with attendant disruption of the autoinhibitory KD-CTD interaction.     

Comparison of the Intrinsic Dynamics of Aminoacyl-tRNA Synthetases

Aminoacyl-tRNA synthetases (AARSs) are an important family of enzymes that catalyze tRNA aminoacylation reaction (Ibba and Soll in Annu Rev Biochem 2000, 69:617–650) [1]. AARSs are grouped into two broad classes (class I and II) based on sequence/structural homology and mode of their interactions with the tRNA molecule (Ibba and Soll in Annu Rev Biochem 2000, 69:617–650) [1]. As protein dynamics play an important role in enzyme function, we explored the intrinsic dynamics of these enzymes using normal mode analysis and investigated if the two classes and six subclasses (Ia–c and IIa–c) of AARSs exhibit any distinct patterns of motion. The present study found that the intrinsic dynamics-based classification of these enzymes is similar to that obtained based on sequence/structural homology for most enzymes. However, the classification of seryl-tRNA synthetase was not straightforward; the internal mobility patterns of this enzyme are comparable to both IIa and IIb AARSs. This study revealed only a few general mobility patterns in these enzymes—(1) the insertion domain is generally engaged in anticorrelated motion with respect to the catalytic domain for both classes of AARSs and (2) anticodon binding domain dynamics are partly correlated and partly anticorrelated with respect to other domains for class I enzymes. In most of the class II AARSs, the anticodon binding domain is predominately engaged in anticorrelated motion with respect to the catalytic domain and correlated to the insertion domain. This study supports the notion that dynamic-based classification could be useful for functional classification of proteins.     

Domain analysis of α-actinin reveals new aspects of its association with F-actin during cytokinesis

α-actinin is a rod-shaped actin cross-linking protein composed of actin binding domain, spectrin-like repeats of the central rod domain and the EF-hand domain. Cytokinesis in mammalian cells involves remodeling of equatorial actin filaments (F-actin) mediated by α-actinin. However, it remains unknown how α-actinin interacts with F-actin at the cleavage furrow. To address this question, we have conducted functional analysis of the mutant that either lacks the ability to cross-link F-actin (ABD) or to bind to F-actin (ΔABD). We found that equatorial localization of α-actinin requires both its F-actin binding and cross-linking activities. Unexpectedly, we also found that overexpression of ΔABD-GFP but not ABD-GFP frequently caused accelerated cytokinesis and ectopic furrowing similar to those observed in cells depleted of α-actinin. Immunofluorescence revealed that overexpression of ΔABD-GFP caused displacement of endogenous α-actinin and a decrease in the density of F-actin throughout the entire cortex. Biochemical experiments showed that ΔABD was able to form heterodimers with endogenous α-actinin. These results suggest that the central rod spectrin-like repeats of α-actinin is sufficient for its dimerization in vivo. Our findings uncover previously unappreciated functions of the α-actinin domains in a cell.     

Crystal structure of a family 54 alpha-L-arabinofuranosidase reveals a novel carbohydrate-binding module that can bind arabinose

As the first known structures of a glycoside hydrolase family 54 (GH54) enzyme, we determined the crystal structures of free and arabinose-complex forms of Aspergillus kawachii IFO4308 alpha-l-arabinofuranosidase (AkAbfB). AkAbfB comprises two domains: a catalytic domain and an arabinose-binding domain (ABD). The catalytic domain has a beta-sandwich fold similar to those of clan-B glycoside hydrolases. ABD has a beta-trefoil fold similar to that of carbohydrate-binding module (CBM) family 13. However, ABD shows a number of characteristics distinctive from those of CBM family 13, suggesting that it could be classified into a new CBM family. In the arabinose-complex structure, one of three arabinofuranose molecules is bound to the catalytic domain through many interactions. Interestingly, a disulfide bond formed between two adjacent cysteine residues recognized the arabinofuranose molecule in the active site. From the location of this arabinofuranose and the results of a mutational study, the nucleophile and acid/base residues were determined to be Glu(221) and Asp(297), respectively. The other two arabinofuranose molecules are bound to ABD. The O-1 atoms of the two arabinofuranose molecules bound at ABD are both pointed toward the solvent, indicating that these sites can both accommodate an arabinofuranose side-chain moiety linked to decorated arabinoxylans.     

Symmetric Assembly of a Decameric Subcomplex in Human Multi-tRNA Synthetase Complex Via Interactions between Glutathione Transferase-Homology Domains and Aspartyl-tRNA Synthetase

Aminoacyl-tRNA synthetases (AARSs) ligate amino acids to their cognate tRNAs during protein synthesis. In humans, eight AARSs and three non-enzymatic AARS-interacting multifunctional proteins (AIMP1–3), which are involved in various biological processes, form a multi-tRNA synthetase complex (MSC). Elucidation of the structures and multiple functions of individual AARSs and AIMPs has aided current understanding of the structural arrangement of MSC components and their assembly processes. Here, we report the crystal structure of a complex comprising a motif from aspartyl-tRNA synthetase (DRS) and the glutathione transferase (GST)-homology domains of methionyl-tRNA synthetase (MRS), glutamyl-prolyl-tRNA synthetase (EPRS), AIMP2, and AIMP3. In the crystal structure, the four GST domains are assembled in the order of MRS-AIMP3-EPRS-AIMP2, and the GST domain of AIMP2 binds DRS through the β-sheet in the GST domain. The C-terminus of AIMP3 enhances the binding of DRS to the tetrameric GST complex. A DRS dimer and two GST tetramers binding to the dimer with 2-fold symmetry complete a decameric complex. The formation of this complex enhances the stability of DRS and enables it to retain its reaction intermediate, aspartyl adenylate. Since the catalytic domains of MRS and EPRS are connected to the decameric complex through their flexible linker peptides, and lysyl-tRNA synthetase and AIMP1 are also linked to the complex via the N-terminal region of AIMP2, the DRS–GST tetramer complex functions as a frame in the MSC.     

Structural and functional roles of deamidation and/or truncation of N- or C-termini in human alpha A-crystallin

The purpose of the study was to compare the effects of deamidation alone, truncation alone, or both truncation and deamidation on structural and functional properties of human lens alphaA-crystallin. Specifically, the study investigated whether deamidation of one or two sites in alphaA-crystallin (i.e., alphaA-N101D, alphaA-N123D, alphaA-N101/123D) and/or truncation of the N-terminal domain (residues 1-63) or C-terminal extension (residues 140-173) affected the structural and functional properties relative to wild-type (WT) alphaA. Human WT-alphaA and human deamidated alphaA (alphaA-N101D, alphaA-N123D, alphaA-N101/123D) were used as templates to generate the following eight N-terminal domain (residues 1-63) deleted or C-terminal extension (residues 140-173) deleted alphaA mutants and deamidated plus N-terminal domain or C-terminal extension deleted mutants: (i) alphaA-NT (NT, N-terminal domain deleted), (ii) alphaA-N101D-NT, (iii) alphaA-N123D-NT, (iv) alphaA-N101/123D-NT, (v) alphaA-CT (CT, C-terminal extension deleted), (vi) alphaA-N101D-CT, (vii) alphaA-N123D-CT, and (viii) alphaA-N101/123D-CT. All of the proteins were purified and their structural and functional (chaperone activity) properties determined. The desired deletions in the alphaA-crystallin mutants were confirmed by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometric analysis. Relative to WT-alphaA homomers, the mutant proteins exhibited major structural and functional changes. The maximum decrease in chaperone activity in homomers occurred on deamidation of N123 residue, but it was substantially restored after N- or C-terminal truncations in this mutant protein. Far-UV circular dichroism (CD) spectral analyses generally showed an increase in the beta-contents in alphaA mutants with deletions of N-terminal domain or C-terminal extension and also with deamidation plus above N- or C-terminal deletions. Intrinsic tryptophan (Trp) and total fluorescence spectral studies suggested altered microenvironments in the alphaA mutant proteins. Similarly, the ANS (8-anilino-1-naphthalenesulfate) binding showed generally increased fluorescence with blue shift on deletion of the N-terminal domain in the deamidated mutant proteins, but opposite effects were observed on deletion of the C-terminal extension. Molecular mass, polydispersity of homomers, and the rate of subunit exchange with WT-alphaB-crystallin increased on deletion of the C-terminal extension in the deamidated alphaA mutants, but on N-terminal domain deletion these values showed variable results based on the deamidation site. In summary, the data suggested that the deamidation alone showed greater effect on chaperone activity than the deletion of N-terminal domain or C-terminal extension of alphaA-crystallin. The N123 residue of alphaA-crystallin plays a crucial role in maintaining its chaperone function. However, both the N-terminal domain and C-terminal extension are also important for the chaperone activity of alphaA-crystallin because the activity was partially or fully recovered following either deletion in the alphaA-N123D mutant. The results of subunit exchange rates among alphaA mutants and WT-alphaB suggested that such exchange is an important determinant in maintenance of chaperone activity following deamidation and/or deletion of the N-terminal domain or C-terminal extension in alphaA-crystallin.     

The carboxy-terminal third of dystrophin enhances actin binding activity

Dystrophin is an actin binding protein that is thought to stabilize the cardiac and skeletal muscle cell membranes during contraction. Here, we investigated the contributions of each dystrophin domain to actin binding function. Cosedimentation assays and pyrene-actin fluorescence experiments confirmed that a fragment spanning two-thirds of the dystrophin molecule [from N-terminal actin binding domain (ABD) 1 through ABD2] bound actin filaments with high affinity and protected filaments from forced depolymerization, but was less effective in both assays than full-length dystrophin. While a construct encoding the C-terminal third of dystrophin displayed no specific actin binding activity or competition with full-length dystrophin, our data show that it confers an unexpected regulation of actin binding by the N-terminal two-thirds of dystrophin when present in cis. Time-resolved phosphorescence anisotropy experiments demonstrated that the presence of the C-terminal third of dystrophin in cis also influences actin interaction by restricting actin rotational amplitude. We propose that the C-terminal region of dystrophin allosterically stabilizes an optimal actin binding conformation of dystrophin.     

The extended loop of the C-terminal carbohydrate-recognition domain of Manduca sexta immulectin-2 is important for ligand binding and functions

Our previous research showed that immulectin-2 (IML-2), a C-type lectin from the tobacco hornworn, Manduca sexta, is a pattern recognition receptor (PRR) that can bind to pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharide (LPS), peptidoglycan (PG) and β-1,3-glucan, and IML-2 plays an important role in cellular encapsulation, melanization, phagocytosis, and prophenoloxidase (proPO) activation. Unlike most mammalian C-type lectins that contain a single carbohydrate-recognition domain (CRD), IML-2 is composed of tandem CRDs, and the C-terminal CRD2 contains an extended loop, which is not present in most C-type CRDs. We hypothesize that the extended loop may participate in ligand binding, encapsulation, melanization, phagocytosis and/or proPO activation in M. sexta. To test this hypothesis, two deletion mutant proteins (IML-2Δ220-244 and IML-2Δ220-257), in which the extended loop of the CRD2 was partially or completely deleted, were expressed and purified. By comparing the characteristics of recombinant IML-2, IML-2Δ220-244 and IML-2Δ220-257, we found that deletion of the extended loop in CRD2 impaired the ability of IML-2 to bind microbial PAMPs and to stimulate proPO activation, indicating that the extended loop of IML-2 plays an important role in ligand binding and biological functions.     

Structural basis for the dynamics of human methionyl-tRNA synthetase in multi-tRNA synthetase complexes

In mammals, eight aminoacyl-tRNA synthetases (AARSs) and three AARS-interacting multifunctional proteins (AIMPs) form a multi-tRNA synthetase complex (MSC). MSC components possess extension peptides for MSC assembly and specific functions. Human cytosolic methionyl-tRNA synthetase (MRS) has appended peptides at both termini of the catalytic main body. The N-terminal extension includes a glutathione transferase (GST) domain responsible for interacting with AIMP3, and a long linker peptide between the GST and catalytic domains. Herein, we determined crystal structures of the human MRS catalytic main body, and the complex of the GST domain and AIMP3. The structures reveal human-specific structural details of the MRS, and provide a dynamic model for MRS at the level of domain orientation. A movement of zinc knuckles inserted in the catalytic domain is required for MRS catalytic activity. Depending on the position of the GST domain relative to the catalytic main body, MRS can either block or present its tRNA binding site. Since MRS is part of a huge MSC, we propose a dynamic switching between two possible MRS conformations; a closed conformation in which the catalytic domain is compactly attached to the MSC, and an open conformation with a free catalytic domain dissociated from other MSC components.     

Terminal amino acids disturb xylanase thermostability and activity

Protein structure is composed of regular secondary structural elements (α-helix and β-strand) and non-regular region. Unlike the helix and strand, the non-regular region consists of an amino acid defined as a disordered residue (DR). When compared with the effect of the helix and strand, the effect of the DR on enzyme structure and function is elusive. An Aspergillus niger GH10 xylanase (Xyn) was selected as a model molecule of (β/α)(8) because the general structure consists of ~10% enzymes. The Xyn has five N-terminal DRs and one C-terminal DR, respectively, which were deleted to construct three mutants, XynΔN, XynΔC, and XynΔNC. Each mutant was ~2-, 3-, or 4-fold more thermostable and 7-, 4-, or 4-fold more active than the Xyn. The N-terminal deletion decreased the xylanase temperature optimum for activity (T(opt)) 6 °C, but the C-terminal deletion increased its T(opt) 6 °C. The N- and C-terminal deletions had opposing effects on the enzyme T(opt) but had additive effects on its thermostability. The five N-terminal DR deletions had more effect on the enzyme kinetics but less effect on its thermo property than the one C-terminal DR deletion. CD data showed that the terminal DR deletions increased regular secondary structural contents, and hence, led to slow decreased Gibbs free energy changes (ΔG(0)) in the thermal denaturation process, which ultimately enhanced enzyme thermostabilities.     

Structural basis for ESCRT-III CHMP3 recruitment of AMSH

Endosomal sorting complexes required for transport (ESCRT) recognize ubiquitinated cargo and catalyze diverse budding processes including multivesicular body biogenesis, enveloped virus egress, and cytokinesis. We present the crystal structure of an N-terminal fragment of the deubiquitinating enzyme AMSH (AMSHΔC) in complex with the C-terminal region of ESCRT-III CHMP3 (CHMP3ΔN). AMSHΔC folds into an elongated 90?? long helical assembly that includes an unusual MIT domain. CHMP3ΔN is unstructured in solution and helical in complex with AMSHΔC, revealing a novel MIT domain interacting motif (MIM) that does not overlap with the CHMP1-AMSH binding site. ITC and SPR measurements demonstrate an unusual high-affinity MIM-MIT interaction. Structural analysis suggests a regulatory role for the N-terminal helical segment of AMSHΔC and its destabilization leads to a loss of function during HIV-1 budding. Our results indicate a tight coupling of ESCRT-III CHMP3 and AMSH functions and provide insight into the regulation of ESCRT-III.     

Crystal Structures of Trypanosomal Histidyl-tRNA Synthetase Illuminate Differences between Eukaryotic and Prokaryotic Homologs

Crystal structures of histidyl-tRNA synthetase (HisRS) from the eukaryotic parasites Trypanosoma brucei and Trypanosoma cruzi provide a first structural view of a eukaryotic form of this enzyme and reveal differences from bacterial homologs. HisRSs in general contain an extra domain inserted between conserved motifs 2 and 3 of the Class II aminoacyl-tRNA synthetase catalytic core. The current structures show that the three-dimensional topology of this domain is very different in bacterial and archaeal/eukaryotic forms of the enzyme. Comparison of apo and histidine-bound trypanosomal structures indicates substantial active-site rearrangement upon histidine binding but relatively little subsequent rearrangement after reaction of histidine with ATP to form the enzyme's first reaction product, histidyladenylate. The specific residues involved in forming the binding pocket for the adenine moiety differ substantially both from the previously characterized binding site in bacterial structures and from the homologous residues in human HisRSs. The essentiality of the single HisRS gene in T. brucei is shown by a severe depression of parasite growth rate that results from even partial suppression of expression by RNA interference.     

Biophysical studies with AICD-47 reveal unique binding behavior characteristic of an unfolded domain

APP intracellular C-terminal domain (AICD-47), generated upon γ-secretase cleavage of amyloid precursor's protein (APP), bears the signature of a classical intrinsically unstructured domain (IUD). Comparing the recent crystal structures of AICD-47 peptides bound to its different adaptors such as protein-tyrosine-binding domain-2 (PTB2) of Fe65 and Src homology 2 (SH2) domain of growth factor receptor binding protein 2 (Grb2), the "conformational switching" of AICD-47 becomes evident. In order to understand different binding processes undertaken by this flexible molecule, upon recognizing different interfaces resulting in different 3D conformations, spectroscopic and calorimetric studies have been done. CD spectroscopy has revealed an overall random coil like structure in different pHs while TFE (2'-2'-2'-trifluoro ethanol) and HFIP (Hexa fluoro isopropanol) induced α-helicity to a certain extent. Binding of Tyr phosphorylated AICD-47 ((P)AICD-47) to Grb2-SH2 domain was carried out by a favorable enthalpic change (ΔH=-197.5±6.2kcalmole(-1) at 25°C) and an unfavorable entropic contribution (ΔS=-631calmol(-1)deg(-1) at 25°C). Alternative conformation of AICD-47 in different biological contexts is another remarkable feature of IUDs which presumably has definitive roles in regulating Alzheimer's disease phenotype.     

Two newly identified SipA domains (F1, F2) steer effector protein localization and contribute to Salmonella host cell manipulation

Salmonella Typhimurium causes bacterial enterocolitis. The type III secretion system (TTSS)-1 is a key virulence determinant of S. Typhimurium mediating host cell invasion and acute enterocolitis. The TTSS-1 effector protein SipA is transported into host cells, accumulates in characteristic foci at the bacteria-host cell interface, manipulates signalling and affects virulence. Two functional domains of SipA have previously been characterized: The N-terminal SipA region (amino acids 1-105) mediates TTSS-1 transport and the C-terminal SipA 'actin-binding' domain (ABD; amino acids 446-685) manipulates F-actin assembly. Little is known about the central region of SipA. In a deletion analysis we found that the central SipA region harbours two distinct functional domains, F1 and F2. They are involved in SipA focus formation and host manipulation. The F1 domain (amino acids 170-271) drives SipA focus formation and domain F2 (amino acids 280-394) enhances this process by mediating SipA-SipA interactions. SipA variants lacking the F1-, the F2- or the actin binding domain were attenuated in virulence assays, namely host cell invasion and/or virulence in a mouse model for enterocolitis. Our results show that the newly identified SipA domains have distinct functions. Nevertheless, cooperation between the SipA domains F1, F2 and ABD is required to promote Salmonella virulence.     

HM1.24/BST-2 is constitutively poly-ubiquitinated at the N-terminal amino acid in the cytoplasmic domain

HM1.24 (also known as BST-2, CD317, and Tetherin) is a type II single-pass transmembrane glycoprotein, which traverses membranes using an N-terminal transmembrane helix and is anchored in membrane lipid rafts via a C-terminal glycosylphosphatidylinositol (GPI). HM1.24 plays a role in diverse cellular functions, including cell signaling, immune modulation, and malignancy. In addition, it also functions as an interferon-induced cellular antiviral restriction factor that inhibits the replication and release of diverse enveloped viruses, and which is counteracted by Vpu, an HIV-1 accessory protein. Vpu induces down-regulation and ubiquitin conjugation to the cytoplasmic domain of HM1.24. However, evidence for ubiquitination site(s) of HM1.24 remains controversial. We demonstrated that HM1.24 is constitutively poly-ubiquitinated at the N-terminal cytoplasmic domain, and that the mutation of all potential ubiquitination sites, including serine, threonine, cysteine, and lysine in the cytoplasmic domain of HM1.24, does not affect the ubiquitination of HM1.24. We further demonstrated that although a GPI anchor is necessary and sufficient for HM1.24 antiviral activities and virion-trapping, the deleted mutant of GPI does not influence the ubiquitination of HM1.24. These results suggest that the lipid raft localization of HM1.24 is not a prerequisite for the ubiquitination. Collectively, our findings demonstrate that the ubiquitination of HM1.24 occurs at the N-terminal amino acid in the cytoplasmic domain and indicate that the constitutive ubiquitination machinery of HM1.24 may differ from the Vpu-induced machinery.