Yong Guang Gao

Solution structure of the ubiquitin-associated domain of human BMSC-UbP and its complex with ubiquitin.

Ubiquitin is an important cellular signal that targets proteins for degradation or regulates their functions. The previously identified BMSC‐UbP protein derived from bone marrow stromal cells contains a ubiquitin‐associated (UBA) domain at the C terminus that has been implicated in linking cellular processes and the ubiquitin system. Here, we report the solution NMR structure of the UBA domain of human BMSC‐UbP protein and its complex with ubiquitin. The structure determination was facilitated by using a solubility‐enhancement tag (SET) GB1, immunoglobulin G binding domain 1 of Streptococcal protein G. The results show that BMSC‐UbP UBA domain is primarily comprised of three α‐helices with a hydrophobic patch defined by residues within the C terminus of helix‐1, loop‐1, and helix‐3. The M‐G‐I motif is similar to the M/L‐G‐F/Y motifs conserved in most UBA domains. Chemical shift perturbation study revealed that the UBA domain binds with the conserved five‐stranded β‐sheet of ubiquitin via hydrophobic interactions with the dissociation constant (KD) of ∼17 μM. The structural model of BMSC‐UbP UBA domain complexed with ubiquitin was constructed by chemical shift mapping combined with the program HADDOCK, which is in agreement with the result from mutagenesis studies. In the complex structure, three residues (Met76, Ile78, and Leu99) of BMSC‐UbP UBA form a trident anchoring the domain to the hydrophobic concave surface of ubiquitin defined by residues Leu8, Ile44, His68, and Val70. This complex structure may provide clues for BMSC‐UbP functions and structural insights into the UBA domains of other ubiquitin‐associated proteins that share high sequence homology with BMSC‐UbP UBA domain.

Crystal structures of E. coli CcmG and its mutants reveal key roles of the N-terminal β-sheet and the fingerprint region

CcmG, also designated DsbE, functions as a periplasmic protein thiol:disulfide oxidoreductase and is required for cytochrome c maturation. Here we report the crystal structures of Escherichia coli CcmG and its two mutants, P144A and the N‐terminal fifty seven‐residue deletion mutant, and two additional deletion mutants were studied by circular dichroism. Structural comparison of E. coli CcmG with its deletion mutants reveals that the N‐terminal β‐sheet is essential for maintaining the folding topology and consequently maintaining the active‐site structure of CcmG. Pro144 and Glu145 are key residues of the fingerprint region of CcmG. Pro144 is in cis‐configuration, and it makes van der Waals interactions with the active‐site disulfide Cys80–Cys83 and forms a CH…O hydrogen bond with Thr82, helping stabilize the active‐site structure. Glu145 forms a salt‐bridge and hydrogen‐bond network with other residues of the fingerprint region and with Arg158, further stabilizing the active‐site structure. The cis‐configuration of Pro144 makes the backbone nitrogen and oxygen of Ala143 exposed to solvent, favorable for interacting with binding partners. The key role of cis‐Pro144 is verified by the P144A mutant, which contains trans‐Ala144 and displays redox property changes. Structural comparison of E. coli CcmG with the recently reported structure of CcmG in complex with the N‐terminal domain of DsbD reveals that Tyr141 undergoes conformational changes upon binding DsbD. A cis‐proline located at the N‐terminus of the first β‐strand of the ββα motif of the thioredoxin‐like domain is a conserved structural feature of the thioredoxin superfamily. Proteins 2006.

Autoinhibitory structure of the WW domain of HYPB/SETD2 regulates its interaction with the proline-rich region of huntingtin.

Huntingtons disease (HD) is an autosomally dominant neurodegenerative disorder caused by expansion of polyglutamine (polyQ) in the huntingtin (Htt) protein. Htt yeast two-hybrid protein B (HYPB/SETD2), a histone methyltransferase, directly interacts with Htt and is involved in HD pathology. Using NMR techniques, we characterized a polyproline (polyP) stretch at the C terminus of HYPB, which directly interacts with the following WW domain and leads this domain predominantly to be in a closed conformational state. The solution structure shows that the polyP stretch extends from the back and binds to the WW core domain in a typical binding mode. This autoinhibitory structure regulates interaction between the WW domain of HYPB and the proline-rich region (PRR) of Htt, as evidenced by NMR and immunofluorescence techniques. This work provides structural and mechanistic insights into the intramolecular regulation of the WW domain in Htt-interacting partners and will be helpful for understanding the pathology of HD.

Human GLTP: Three distinct functions for the three tryptophans in a novel peripheral amphitropic fold

Human glycolipid transfer protein (GLTP) serves as the GLTP-fold prototype, a novel, to our knowledge, peripheral amphitropic fold and structurally unique lipid binding motif that defines the GLTP superfamily. Despite conservation of all three intrinsic Trps in vertebrate GLTPs, the Trp functional role(s) remains unclear. Herein, the issue is addressed using circular dichroism and fluorescence spectroscopy along with an atypical Trp point mutation strategy. Far-ultraviolet and near-ultraviolet circular dichroism spectroscopic analyses showed that W96F-W142Y-GLTP and W96Y-GLTP retain their native conformation and stability, whereas W85Y-W96F-GLTP is slightly altered, in agreement with relative glycolipid transfer activities of >90%, ∼85%, and ∼45%, respectively. In silico three-dimensional modeling and acrylamide quenching of Trp fluorescence supported a nativelike folding conformation. With the Trp⁹⁶-less mutants, changes in emission intensity, wavelength maximum, lifetime, and time-resolved anisotropy decay induced by phosphoglyceride membranes lacking or containing glycolipid and by excitation at different wavelengths along the absorption-spectrum red edge indicated differing functions for W142 and W85. The data suggest that W142 acts as a shallow-penetration anchor during docking with membrane interfaces, whereas the buried W85 indole helps maintain proper folding and possibly regulates membrane-induced transitioning to a glycolipid-acquiring conformation. The findings illustrate remarkable versatility for Trp, providing three distinct intramolecular functions in the novel amphitropic GLTP fold.

Human Glycolipid Transfer Protein Gene (GLTP) Expression Is Regulated by Sp1 and Sp3 INVOLVEMENT OF THE BIOACTIVE SPHINGOLIPID CERAMIDE

Glycolipid transfer protein (GLTP) accelerates glycolipid intermembrane transfer via a unique lipid transfer/binding fold (GLTP fold) that defines the GLTP superfamily and is the prototype for functional GLTP-like domains in larger proteins, i.e. FAPP2. Human GLTP is encoded by the single-copy GLTP gene on chromosome 12 (12q24.11 locus), but regulation of GLTP gene expression remains completely unexplored. Herein, the ability of glycosphingolipids (and their sphingolipid metabolites) to regulate the transcriptional expression of GLTP via its promoter has been evaluated. Using luciferase and GFP reporters in concert with deletion mutants, the constitutive and basal (225 bp; ∼78% G+C) human GLTP promoters have been defined along with adjacent regulatory elements. Despite high G+C content, translational regulation was not evident by the mammalian target of rapamycin pathway. Four GC-boxes were shown to be functional Sp1/Sp3 transcription factor binding sites. Mutation of one GC-box was particularly detrimental to GLTP transcriptional activity. Sp1/Sp3 RNA silencing and mithramycin A treatment significantly inhibited GLTP promoter activity. Among tested sphingolipid analogs of glucosylceramide, sulfatide, ganglioside GM1, ceramide 1-phosphate, sphingosine 1-phosphate, dihydroceramide, sphingosine, only ceramide, a nonglycosylated precursor metabolite unable to bind to GLTP protein, induced GLTP promoter activity and raised transcript levels in vivo. Ceramide treatment partially blocked promoter activity decreases induced by Sp1/Sp3 knockdown. Ceramide treatment also altered the in vivo binding affinity of Sp1 and Sp3 for the GLTP promoter and decreased Sp3 acetylation. This study represents the first characterization of any Gltp gene promoter and links human GLTP expression to sphingolipid homeostasis through ceramide.

Conformational Folding and Stability of the HET-C2 Glycolipid Transfer Protein Fold: Does a Molten Globule-like State Regulate Activity?

The glycolipid transfer protein (GLTP) superfamily is defined by the human GLTP fold that represents a novel motif for lipid binding and transfer and for reversible interaction with membranes, i.e., peripheral amphitropic proteins. Despite limited sequence homology with human GLTP, we recently showed that HET-C2 GLTP of Podospora anserina is organized conformationally as a GLTP fold. Currently, insights into the folding stability and conformational states that regulate GLTP fold activity are almost nonexistent. To gain such insights into the disulfide-less GLTP fold, we investigated the effect of a change in pH on the fungal HET-C2 GLTP fold by taking advantage of its two tryptophans and four tyrosines (compared to three tryptophans and 10 tyrosines in human GLTP). pH-induced conformational alterations were determined by changes in (i) intrinsic tryptophan fluorescence (intensity, emission wavelength maximum, and anisotropy), (ii) circular dichroism over the near-UV and far-UV ranges, including thermal stability profiles of the derivatized molar ellipticity at 222 nm, (iii) fluorescence properties of 1-anilinonaphthalene-8-sulfonic acid, and (iv) glycolipid intermembrane transfer activity monitored by Förster resonance energy transfer. Analyses of our recently determined crystallographic structure of HET-C2 (1.9 Å) allowed identification of side chain electrostatic interactions that contribute to HET-C2 GLTP fold stability and can be altered by a change in pH. Side chain interactions include numerous salt bridges and interchain cation-π interactions, but not intramolecular disulfide bridges. Histidine residues are especially important for stabilizing the local positioning of the two tryptophan residues and the conformation of adjacent chains. Induction of a low-pH-induced, molten globule-like state inhibited glycolipid intermembrane transfer by the HET-C2 GLTP fold.

Structural Insights into the Interaction between a Potent Anti-inflammatory Protein, Viral CC Chemokine Inhibitor (vCCI), and the Human CC Chemokine, Eotaxin-1

Background: The mechanism used by viral protein vCCI to tightly bind to many CC chemokines is not known. Results: Specific positively charged residues in the chemokine eotaxin-1 mediate binding to vCCI. Conclusion: Basic residues in the chemokine each provide incremental affinity for vCCI. Significance: This work shows how vCCI can bind a variety of CC chemokines. Chemokines play important roles in the immune system, not only recruiting leukocytes to the site of infection and inflammation but also guiding cell homing and cell development. The soluble poxvirus-encoded protein viral CC chemokine inhibitor (vCCI), a CC chemokine inhibitor, can bind to human CC chemokines tightly to impair the host immune defense. This protein has no known homologs in eukaryotes and may represent a potent method to stop inflammation. Previously, our structure of the vCCI·MIP-1β (macrophage inflammatory protein-1β) complex indicated that vCCI uses negatively charged residues in β-sheet II to interact with positively charged residues in the MIP-1β N terminus, 20s region and 40s loop. However, the interactions between vCCI and other CC chemokines have not yet been fully explored. Here, we used NMR and fluorescence anisotropy to study the interaction between vCCI and eotaxin-1 (CCL11), a CC chemokine that is an important factor in the asthma response. NMR results reveal that the binding pattern is very similar to the vCCI·MIP-1β complex and suggest that electrostatic interactions provide a major contribution to binding. Fluorescence anisotropy results on variants of eotaxin-1 further confirm the critical roles of the charged residues in eotaxin-1. In addition, the binding affinity between vCCI and other wild type CC chemokines, MCP-1 (monocyte chemoattractant protein-1), MIP-1β, and RANTES (regulated on activation normal T cell expressed and secreted), were determined as 1.1, 1.2, and 0.22 nm, respectively. To our knowledge, this is the first work quantitatively measuring the binding affinity between vCCI and multiple CC chemokines.

Cytoplasmic Ubiquitin-Specific Protease 19 (USP19) Modulates Aggregation of Polyglutamine-Expanded Ataxin-3 and Huntingtin through the HSP90 Chaperone

Ubiquitin-specific protease 19 (USP19) is one of the deubiquitinating enzymes (DUBs) involved in regulating the ubiquitination status of substrate proteins. There are two major isoforms of USP19 with distinct C-termini; the USP19_a isoform has a transmembrane domain for anchoring to the endoplasmic reticulum, while USP19_b contains an EEVD motif. Here, we report that the cytoplasmic isoform USP19_b up-regulates the protein levels of the polyglutamine (polyQ)-containing proteins, ataxin-3 (Atx3) and huntingtin (Htt), and thus promotes aggregation of their polyQ-expanded species in cell models. Our data demonstrate that USP19_b may orchestrate the stability, aggregation and degradation of the polyQ-expanded proteins through the heat shock protein 90 (HSP90) chaperone system. USP19_b directly interacts with HSP90 through its N-terminal CS (CHORD and SGT1)/P23 domains. In conjunction with HSP90, the cytoplasmic USP19 may play a key role in triage decision for the disease-related polyQ-expanded substrates, suggesting a function of USP19 in quality control of misfolded proteins by regulating their protein levels.

Solution structure of the ubiquitin‐like domain of human DC‐UbP from dendritic cells

The previously identified dendritic cell‐derived ubiquitin‐like protein (DC‐UbP) was implicated in cellular differentiation and apoptosis. Sequence alignment suggested that it contains a ubiquitin‐like (UbL) domain in the C terminus. Here, we present the solution NMR structure and backbone dynamics of the UbL domain of DC‐UbP. The overall structure of the domain is very similar to that of Ub despite low similarity (<30%) in amino‐acid sequence. One distinct feature of the domain structure is its highly positively charged surface that is different from the corresponding surfaces of the well‐known UbL modifiers, Ub, NEDD8, and SUMO‐1. The key amino‐acid residues responsible for guiding polyubiquitinated proteins to proteasome degradation in Ub are not conserved in the UbL domain. This implies that the UbL domain of DC‐UbP may have its own specific interaction partners with other yet unknown cellular functions related to the Ub pathway.

Human glycolipid transfer protein (GLTP) expression modulates cell shape

Glycolipid transfer protein (GLTP) accelerates glycosphingolipid (GSL) intermembrane transfer via a unique lipid transfer/binding fold (GLTP-fold) that defines the GLTP superfamily and is the prototype for GLTP-like domains in larger proteins, i.e. phosphoinositol 4-phosphate adaptor protein-2 (FAPP2). Although GLTP-folds are known to play roles in the nonvesicular intracellular trafficking of glycolipids, their ability to alter cell phenotype remains unexplored. In the present study, overexpression of human glycolipid transfer protein (GLTP) was found to dramatically alter cell phenotype, with cells becoming round between 24 and 48 h after transfection. By 48 h post transfection, ∼70% conversion to the markedly round shape was evident in HeLa and HEK-293 cells, but not in A549 cells. In contrast, overexpression of W96A-GLTP, a liganding-site point mutant with abrogated ability to transfer glycolipid, did not alter cell shape. The round adherent cells exhibited diminished motility in wound healing assays and an inability to endocytose cholera toxin but remained viable and showed little increase in apoptosis as assessed by poly(ADP-ribose) polymerase cleavage. A round cell phenotype also was induced by overexpression of FAPP2, which binds/transfers glycolipid via its C-terminal GLTP-like fold, but not by a plant GLTP ortholog (ACD11), which is incapable of glycolipid binding/transfer. Screening for human protein partners of GLTP by yeast two hybrid screening and by immuno-pulldown analyses revealed regulation of the GLTP-induced cell rounding response by interaction with δ-catenin. Remarkably, while δ-catenin overexpression alone induced dendritic outgrowths, coexpression of GLTP along with δ-catenin accelerated transition to the rounded phenotype. The findings represent the first known phenotypic changes triggered by GLTP overexpression and regulated by direct interaction with a p120-catenin protein family member.