Tollip and Tom1 form a complex and recruit ubiquitin-conjugated proteins onto early endosomes

Tom1 (target of Myb1) is a protein of unknown function. Tom1 and its relative Tom1L1 have an N-terminal VHS (Vps27p/Hrs/Stam) domain followed by a GAT (GGA and Tom1) domain, both of which are also found in the GGA (Golgi-localizing, gamma-adaptin ear domain homology, ADP-ribosylation factor-binding protein) family of proteins. Although the VHS and GAT domains of GGA proteins bind to transmembrane cargo proteins and the small GTPase ADP-ribosylation factor, respectively, the VHS and GAT domains of Tom1 are unable to interact with these proteins. In this study, we show that the GAT domains of Tom1 and Tom1L1 interact with ubiquitin and Tollip (Toll-interacting protein). Ubiquitin bound the GAT domains of Tom1, Tom1L1, and GGA proteins, whereas Tollip interacted specifically with Tom1 and Tom1L1. Ubiquitin and Tollip bound to an overlapping region of the Tom1-GAT domain in a mutually exclusive manner. Tom1 was predominantly cytosolic when expressed in cells. On the other hand, Tollip was localized on early endosomes and recruited Tom1 and ubiquitinated proteins. These observations suggest that Tollip and Tom1 form a complex and regulate endosomal trafficking of ubiquitinated proteins.     

The Emerging Role of VHS Domain‐Containing Tom1, Tom1L1 and Tom1L2 in Membrane Trafficking

The maintenance of cellular homeostasis and execution of regulatory mechanisms to dynamically govern various cellular processes require the correct delivery of proteins to their target subcellular compartments. It is estimated that over 30% of the proteins encoded by the human genome, projected to encode about 25 000 proteins and other macromolecules, are delivered to the secretory and endocytic pathways where movement of proteins between various compartments is primarily mediated by vesicles/carriers budding from one compartment for delivery to another. Sorting of cargo proteins into budding vesicles/carriers is mediated by adaptors that link the cargo proteins to the coat proteins. The adaptor function of VHS domain proteins, GGA proteins, STAM proteins and Hrs is well‐established and is evolutionarily conserved from yeast to humans. Recent studies suggest that Tom1, Tom1L1 and Tom1L2 subfamily of VHS domain proteins, which do not exist in yeast, are emerging as novel regulators for post‐Golgi trafficking and signaling.     

Backbone 1H, 13C, and 15N assignments for the tandem ubiquitin binding domains of signal transducing adapter molecule 1

Signal transducing adapter molecule (STAM) forms the endosomal sorting complex required for transport-0 (ESCRT-0) complex with hepatocyte growth factor-regulated substrate (Hrs) to sort the ubiquitinated cargo proteins from the early endosomes to the ESCRT-1 complex. ESCRT-0 complex, STAM and Hrs, contains multiple ubiquitin binding domains, in which STAM has two ubiquitin binding domains, Vps27/Hrs/Stam (VHS) and ubiquitin interacting motif (UIM) at its N-terminus. By the cooperation of the multiple ubiquitin binding domains, the ESCRT-0 complex recognizes poly-ubiquitin, especially Lys63-linked ubiquitin. Here, we report the backbone resonance assignments and the secondary structure of the N-terminal 191 amino acids of the human STAM1 which includes the VHS domain and UIM. The {¹H}-¹⁵N heteronuclear NOE experiments revealed that an unstructured and flexible loop region connects the VHS domain and UIM. Our work provides the basic information for the further NMR investigation of the interaction between STAM1 and poly-ubiquitin.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N backbone and side-chain resonance assignments of the ZnF-UBP domain of USP20/VDU2

Deubiquitinase USP20/VDU2 has been identified as a regulator of multiple proteins including hypoxia-inducible factor (HIF)-1α, β2-adrenergic receptor, and tumor necrosis factor receptor associated factor 6 etc. It contains four structural domains, including an N-terminal zinc-finger ubiquitin binding domain (ZnF-UBP) that potentially helps USP20 to recruit its ubiquitin substrates. Here we report the ¹H, ¹³C and ¹⁵N backbone and side-chain resonance assignments of the ZnF-UBP domain of USP20/VDU2. The BMRB accession number is 26901. The secondary structural elements predicted from the NMR data reveal a global fold consisting of three α-helices and four β-strands. The complete assignments can be used to explore the protein dynamics of the USP20 ZnF-UBP and its interactions with monoubiquitin and ubiquitin chains.     

<Superscript>1</Superscript>H, <Superscript>15</Superscript>N, <Superscript>13</Superscript>C resonance assignments for <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> Rad23 UBL domain

Rad23 functions in nucleotide excision repair and proteasome-mediated protein degradation. It has four distinct structural domains that are connected by flexible linker regions, including an N-terminal ubiquitin-like (UBL) domain that binds proteasomes. We report in this NMR study the ¹H, ¹⁵N and ¹³C resonance assignments for the backbone and side chain atoms of the Rad23 UBL domain (Rad23^UBL) with BioMagResBank accession number 25825. We find that a Rad23 proline amino acid (P20) located in a loop undergoes isomerization. The secondary structural elements predicted from the NMR data fit well to that of the Rad23^UBL when complexed with E4 ubiquitin ligase Ufd2, as reported in a crystallographic structure. These complete assignments can be used to study the protein dynamics of the Rad23^UBL and its interaction of with other ubiquitin receptors or proteasome subunits.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N resonance assignments for a chemokine receptor-binding domain of FROUNT,a cytoplasmic regulator of chemotaxis

FROUNT is a cytoplasmic protein that interacts with the membrane-proximal C-terminal regions (Pro-Cs) of the CCR2 and CCR5 chemokine receptors. The interactions between FROUNT and the chemokine receptors play an important role in the migration of inflammatory immune cells. Therefore, FROUNT is a potential drug target for inflammatory diseases. However, the structural basis of the interactions between FROUNT and the chemokine receptors remains to be elucidated. We previously identified the C-terminal region (residues 532–656) of FROUNT as the structural domain responsible for the Pro-C binding, referred to as the chemokine receptor-binding domain (CRBD), and then constructed its mutant, bearing L538E/P612S mutations, with improved NMR spectral quality, referred to as CRBD_LEPS. We now report the main-chain and side-chain ¹H, ¹³C, and ¹⁵N resonance assignments of CRBD_LEPS. The NMR signals of CRBD_LEPS were well dispersed and their intensities were uniform on the ¹H–¹⁵N HSQC spectrum, and thus almost all of the main-chain and side-chain resonances were assigned. This assignment information provides the foundation for NMR studies of the three-dimensional structure of CRBD_LEPS in solution and its interactions with chemokine receptors.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N NMR assignments of a bacterial immunoglobulin-like domain (group 2) of a protein of a bacterium <Emphasis Type="Italic">Paenarthrobacter aurescens</Emphasis> TC1

The bacterial immunoglobulin-like (Big) domain is one of the prevalent domain types, which facilitates cell–cell adhesion by assembling into multi-domain architectures. We selected a four Big_2 domain protein (named ‘Arig’) from a Gram positive, Paenarthrobacter aurescens TC1 (known earlier as Arthrobacter aurescens TC1). In an attempt to characterize structural and ligand-binding features of individual Big_2 domains, we have cloned, overexpressed, isolated and purified the second Big_2 domain of Arig along with a few of its adjacent Big_2 domain residues (residue 143 to 269) referred to as ‘Arig2’. The ¹³C/¹⁵N-doubly-labeled His-tagged Arig2 (133 residues long) showed an ordered conformation as revealed by the well dispersed 2D [¹⁵N-¹H]-HSQC spectrum. Subsequently, a suite of heteronuclear 3D NMR experiments has enabled almost complete ¹H, ¹³C and ¹⁵N NMR resonance assignments of Arig2.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C,and <Superscript>15</Superscript>N resonance assignments of FAS1-IV domain of human periostin,a component of extracellular matrix proteins

Periostin, an extracellular matrix protein, is secreted by fibroblasts and is overexpressed in various types of cancers. The four internal repeat fasciclin 1 (FAS1) domains of human periostin play crucial roles in promoting tumor metastasis and progression via interaction with cell surface integrins. Among four FAS1 domains of human periostin, the fourth FAS1 domain (FAS1-IV) was prepared for NMR study, since only FAS1-IV was highly soluble, and showed a well-dispersed 2D ¹H-¹⁵N HSQC spectrum. Here, we report nearly complete backbone and side chain resonance assignments and a secondary structural analysis of the FAS1-IV domain as first steps toward the structure determination of FAS1-IV of human periostin.     

Dictyostelium Tom1 Participates to an Ancestral ESCRT-0 Complex

Sorting of ubiquitinated proteins to multivesicular bodies (MVBs) in mammalian cells relies on proteins with a Vps27/Hrs/STAM (VHS) domain. Here, we show that the amoeba Dictyostelium presents only one protein with a VHS domain: DdTom1. We demonstrate that the VHS domain of DdTom1 is followed by a Golgi-localized, γ-ear-containing, ADP-ribosylation-factor-binding and Tom1 (GAT) domain that binds ubiquitin, and by a non-conserved C-terminal domain that can recruit clathrin, EGFr pathway substrate 15 and tumor susceptibility gene 101, a component of the MVB biogenesis machinery [endosomal complexes required for transport (ESCRT) complexes]. Both VHS and GAT domains interact with phospholipids and therefore could ensure the recruitment of DdTom1 to endosomal membranes. We propose that DdTom1 participates in an ancestral ESCRT-0 complex implicated in the sorting of ubiquitinated proteins into MVBs.     

STAM proteins bind ubiquitinated proteins on the early endosome via the VHS domain and ubiquitin-interacting motif

Conjugation with ubiquitin acts as a sorting signal for proteins in the endocytic and biosynthetic pathways at the endosome. Signal-transducing adaptor molecule (STAM) proteins, STAM1 and STAM2, are associated with hepatocyte growth factor-regulated substrate (Hrs) but their function remains unknown. Herein, we show that STAM proteins bind ubiquitin and ubiquitinated proteins and that the tandemly located VHS (Vps27/Hrs/STAM) domain and ubiquitin-interacting motif serve as the binding site(s). STAM proteins colocalize with Hrs on the early endosome. Overexpression of STAM proteins, but not their mutants lacking the ubiquitin-binding activity, causes the accumulation of ubiquitinated proteins and ligand-activated epidermal growth factor receptor on the early endosome. These results suggest that through interaction with ubiquitinated cargo proteins on the early endosome via the VHS domain and ubiquitin-interacting motif, STAM proteins participate in the sorting of cargo proteins for trafficking to the lysosome.     

Interactions of TOM1L1 with the multivesicular body sorting machinery

Tom1L1 (Tom1-like1) and related proteins Tom1 (Target of Myb1) and Tom1L2 (Tom1-like2) constitute a new protein family characterized by the presence of a VHS (Vps27p/Hrs/Stam) domain in the N-terminal portion followed by a GAT (GGA and Tom) domain. Recently it was demonstrated that the GAT domain of both Tom1 and Tom1L1 binds ubiquitin, suggesting that these proteins might participate in the sorting of ubiquitinated proteins into multivesicular bodies (MVBs). Here we report a novel interaction between Tom1L1 and members of the MVB sorting machinery. Specifically, we found that the VHS domain of Tom1L1 interacts with Hrs (Hepatocyte growth factor-regulated tyrosine kinase substrate), whereas a PTAP motif, located between the VHS and GAT domain of Tom1L1, is responsible for binding to TSG101 (tumor susceptibility gene 101). Myc epitope-tagged Tom1L1 showed a cytosolic distribution but was recruited to endosomes following Hrs expression. In addition, Tom1L1 possesses several tyrosine motifs at the C-terminal region that mediate interactions with members of the Src family kinases and other signaling proteins such as Grb2 and p85. We showed that a fraction of Fyn kinase localizes at endosomes and that this distribution becomes more evident after epidermal growth factor internalization. Moreover, expression of a constitutive active form of Fyn also promoted the recruitment of Tom1L1 to enlarged endosomes. Taken together, we propose that Tom1L1 could act as an intermediary between signaling and degradative pathways.     

<Superscript>1</Superscript>H, <Superscript>15</Superscript>N, <Superscript>13</Superscript>C resonance assignments for pyrazinoic acid binding domain of ribosomal protein S1 from <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

Ribosomal protein S1 of Mycobacterium tuberculosis (MtRpsA) binds to ribosome and mRNA, and plays significant role in the regulation of translation initiation, conventional protein synthesis and transfer-messenger RNA (tmRNA) mediated trans-translation. It has been identified as the target of pyrazinoic acid (POA), a bactericidal moiety from hydrolysis of pyrazinamide, which is a mainstay of combination therapy for tuberculosis. POA prevented the interactions between the C-terminal S1 domain of MtRpsA (residues 280–368, MtRpsA^CTD_S1) and tmRNA; so that POA can inhibit the trans-translation, which is a key component of multiple quality control pathways in bacteria. However, the details of molecular mechanism and dynamic characteristics for MtRpsA^CTD_S1 interactions with POA, tmRNA or mRNA are still unclear. Here we present the ¹H, ¹⁵N, ¹³C resonance assignments of MtRpsA^CTD_S1 as well as the secondary structure information based on backbone chemical shifts, which lay foundation for further solution structure determination, dynamic properties characterization and interactions investigation between MtRpsA^CTD_S1 and tmRNA, RNA or POA.     

Magnetic Resonance of Isotope Hybrid Flavoprotein <Superscript>2</Superscript>H-Flavoprotein (<Superscript>1</Superscript>-Flavin Mononucleotide)

FLAVOPROTEINS lend themselves particularly well to study by the combined techniques of magnetic resonance and isotope substitution^1–3. Recent electron spin resonance (ESR) and electron-nuclear double resonance (ENDOR) investigations have already indicated the utility of this approach to the study of the structure and function of flavin and flavoproteins^4–8. We have now prepared an unusual isotope hybrid flavoprotein that gives promise of great value for magnetic resonance studies. This substance is obtained by the introduction (by exchange) of an ¹H-flavin mononucleotide (¹H-FMN) prosthetic group into (fully deuterated) ²H-flavoprotein that can be isolated from the blue-green thermophilic alga Synechococcus lividus. This isotope hybrid protein makes it possible to apply in a unique way proton magnetic resonance (PMR) and ESR to the chemistry of protein-bound FMN.     

<Superscript>1</Superscript>H, <Superscript>15</Superscript>N, <Superscript>13</Superscript>C backbone resonance assignment of the C-terminal domain of enzyme I from <Emphasis Type="Italic">Thermoanaerobacter tengcongensis</Emphasis>

Phosphoenolpyruvate binding to the C-terminal domain (EIC) of enzyme I of the bacterial phosphotransferase system (PTS) initiates a phosphorylation cascade that results in sugar translocation across the cell membrane and controls a large number of essential pathways in bacterial metabolism. EIC undergoes an expanded to compact conformational equilibrium that is regulated by ligand binding and determines the phosphorylation state of the overall PTS. Here, we report the backbone ¹H, ¹⁵N and ¹³C chemical shift assignments of the 70 kDa EIC dimer from the thermophilic bacterium Thermoanaerobacter tengcongensis. Assignments were obtained at 70 °C by heteronuclear multidimensional NMR spectroscopy. In total, 90% of all backbone resonances were assigned, with 264 out of a possible 299 residues assigned in the ¹H–¹⁵N TROSY spectrum. The secondary structure predicted from the assigned backbone resonance using the program TALOS+ is in good agreement with the X-ray crystal structure of T. tengcongensis EIC. The reported assignments will allow detailed structural and thermodynamic investigations on the coupling between ligand binding and conformational dynamics in EIC.     

Near-complete <Superscript>1</Superscript>H, <Superscript>13</Superscript>C, <Superscript>15</Superscript>N resonance assignments of dimethylsulfoxide-denatured TGFBIp FAS1-4 A546T

The transforming growth factor beta induced protein (TGFBIp) is a major protein component of the human cornea. Mutations occurring in TGFBIp may cause corneal dystrophies, which ultimately lead to loss of vision. The majority of the disease-causing mutations are located in the C-terminal domain of TGFBIp, referred as the fourth fascilin-1 (FAS1-4) domain. In the present study the FAS1-4 Ala546Thr, a mutation that causes lattice corneal dystrophy, was investigated in dimethylsulfoxide using liquid-state NMR spectroscopy, to enable H/D exchange strategies for identification of the core formed in mature fibrils. Isotope-labeled fibrillated FAS1-4 A546T was dissolved in a ternary mixture 95/4/1 v/v/v% dimethylsulfoxide/water/trifluoroacetic acid, to obtain and assign a reference 2D ¹H–¹⁵N HSQC spectrum for the H/D exchange analysis. Here, we report the near-complete assignments of backbone and aliphatic side chain ¹H, ¹³C and ¹⁵N resonances for unfolded FAS1-4 A546T at 25 °C.     

<Superscript>1</Superscript>H, <Superscript>15</Superscript>N,and <Superscript>13</Superscript>C chemical shift assignments of the regulatory domain of human calcineurin

Calcineurin (CaN) plays an important role in T-cell activation, cardiac system development and nervous system function. Previous studies have demonstrated that the regulatory domain (RD) of CaN binds calmodulin (CaM) towards the N-terminal end. Calcium-loaded CaM activates the serine/threonine phosphatase activity of CaN by binding to the RD, although the mechanistic details of this interaction remain unclear. It is thought that CaM binding at the RD displaces the auto-inhibitory domain (AID) from the active site of CaN, activating phosphatase activity. In the absence of calcium-loaded CaM, the RD is disordered, and binding of CaM induces folding in the RD. In order to provide mechanistic detail about the CaM–CaN interaction, we have undertaken an NMR study of the RD of CaN. Complete ¹³C, ¹⁵N and ¹H assignments of the RD of CaN were obtained using solution NMR spectroscopy. The backbone of RD has been assigned using a combination of ¹³C-detected CON-IPAP experiments as well as traditional HNCO, HNCA, HNCOCA and HNCACB-based 3D NMR spectroscopy. A ¹⁵N-resolved TOCSY experiment has been used to assign Hα and Hβ chemical shifts.     

Simultaneous <Superscript>1</Superscript>H- or <Superscript>2</Superscript>H-, <Superscript>15</Superscript>N- and multiple-band-selective <Superscript>13</Superscript>C-decoupling during acquisition in <Superscript>13</Superscript>C-detected experiments with proteins and oligonucleotides

Significant resolution improvement in ¹³C,¹³C-TOCSY spectra of uniformly deuterated and ¹³C, ¹⁵N-labeled protein and ¹³C,¹⁵N-labeled RNA samples is achieved by introduction of multiple-band-selective ¹³C-homodecoupling applied simultaneously with ¹H- or ²H- and ¹⁵N-decoupling at all stages of multidimensional experiments including signal acquisition period. The application of single, double or triple band-selective ¹³C-decoupling in 2D-[¹³C,¹³C]-TOCSY experiments during acquisition strongly simplifies the homonuclear splitting pattern. The technical aspects of complex multiple-band homonuclear decoupling and hardware requirements are discussed. The use of this technique (i) facilitates the resonance assignment process as it reduces signal overlap in homonuclear ¹³C-spectra and (ii) possibly improves the signal-to-noise ratio through multiplet collapse. It can be applied in any ¹³C-detected experiment.     

<Superscript>1</Superscript>H, <Superscript>15</Superscript>N and <Superscript>13</Superscript>C backbone resonance assignments of pentaerythritol tetranitrate reductase from <Emphasis Type="Italic">Enterobacter cloacae</Emphasis> PB2

Pentaerythritol tetranitrate reductase (PETNR) is a flavoenzyme possessing a broad substrate specificity and is a member of the Old Yellow Enzyme family of oxidoreductases. As well as having high potential as an industrial biocatalyst, PETNR is an excellent model system for studying hydrogen transfer reactions. Mechanistic studies performed with PETNR using stopped-flow methods have shown that tunneling contributes towards hydride transfer from the NAD(P)H coenzyme to the flavin mononucleotide (FMN) cofactor and fast protein dynamics have been inferred to facilitate this catalytic step. Herein, we report the near-complete ¹H, ¹⁵N and ¹³C backbone resonance assignments of PETNR in a stoichiometric complex with the FMN cofactor in its native oxidized form, which were obtained using heteronuclear multidimensional NMR spectroscopy. A total of 97% of all backbone resonances were assigned, with 333 out of a possible 344 residues assigned in the ¹H–¹⁵N TROSY spectrum. This is the first report of an NMR structural study of a flavoenzyme from the Old Yellow Enzyme family and it lays the foundation for future investigations of functional dynamics in hydride transfer catalytic mechanism.