Yong Gong

Crystal structure of the neurotrophin-3 and p75NTR symmetrical complex

Neurotrophins (NTs) are important regulators for the survival, differentiation and maintenance of different peripheral and central neurons. NTs bind to two distinct classes of glycosylated receptor: the p75 neurotrophin receptor (p75NTR) and tyrosine kinase receptors (Trks). Whereas p75NTR binds to all NTs, the Trk subtypes are specific for each NT. The question of whether NTs stimulate p75NTR by inducing receptor homodimerization is still under debate. Here we report the 2.6-Å resolution crystal structure of neurotrophin-3 (NT-3) complexed to the ectodomain of glycosylated p75NTR. In contrast to the previously reported asymmetric complex structure, which contains a dimer of nerve growth factor (NGF) bound to a single ectodomain of deglycosylated p75NTR (ref. 3), we show that NT-3 forms a central homodimer around which two glycosylated p75NTR molecules bind symmetrically. Symmetrical binding occurs along the NT-3 interfaces, resulting in a 2:2 ligand–receptor cluster. A comparison of the symmetrical and asymmetric structures reveals significant differences in ligand–receptor interactions and p75NTR conformations. Biochemical experiments indicate that both NT-3 and NGF bind to p75NTR with 2:2 stoichiometry in solution, whereas the 2:1 complexes are the result of artificial deglycosylation. We therefore propose that the symmetrical 2:2 complex reflects a native state of p75NTR activation at the cell surface. These results provide a model for NTs-p75NTR recognition and signal generation, as well as insights into coordination between p75NTR and Trks.

Structural insight into dGTP-dependent activation of tetrameric SAMHD1 deoxynucleoside triphosphate triphosphohydrolase

SAMHD1 is a dGTP-activated deoxynucleoside triphosphate triphosphohydrolase (dNTPase) whose dNTPase activity has been linked to HIV/SIV restriction. The mechanism of its dGTP-activated dNTPase function remains unclear. Recent data also indicate that SAMHD1 regulates retrotransposition of LINE-1 elements. Here we report the 1.8-Å crystal structure of homotetrameric SAMHD1 in complex with the allosteric activator and substrate dGTP/dATP. The structure indicates the mechanism of dGTP-dependent tetramer formation, which requires the cooperation of three subunits and two dGTP/dATP molecules at each allosteric site. Allosteric dGTP binding induces conformational changes at the active site, allowing a more stable interaction with the substrate and explaining the dGTP-induced SAMHD1 dNTPase activity. Mutations of dGTP binding residues in the allosteric site affect tetramer formation, dNTPase activity and HIV-1 restriction. dGTP-triggered tetramer formation is also important for SAMHD1-mediated LINE-1 regulation. The structural and functional information provided here should facilitate future investigation of SAMHD1 function, including dNTPase activity, LINE-1 modulation and HIV-1 restriction.

Structure and Functional Implications of the Human Rad9-Hus1-Rad1 Cell Cycle Checkpoint Complex

Cellular DNA lesions are efficiently countered by DNA repair in conjunction with delays in cell cycle progression. Previous studies have demonstrated that Rad9, Hus1, and Rad1 can form a heterotrimeric complex (the 9-1-1 complex) that plays dual roles in cell cycle checkpoint activation and DNA repair in eukaryotic cells. Although the 9-1-1 complex has been proposed to form a toroidal structure similar to proliferating cell nuclear antigen (PCNA), which plays essential roles in DNA replication and repair, the structural basis by which it performs different functions has not been elucidated. Here we report the crystal structure of the human 9-1-1 complex at 3.2 Å resolution. The crystal structure, together with biochemical assays, reveals that the interdomain connecting loops (IDC loop) of hRad9, hHus1, and hRad1 are largely divergent, and further cocrystallization study indicates that a PCNA-interacting box (PIP box)-containing peptide derived from hFen1 binds tightly to the interdomain connecting loop of hRad1, providing the molecular basis for the damage repair-specific activity of the 9-1-1 complex in contrast to PCNA. Furthermore, structural comparison with PCNA reveals other unique structural features of the 9-1-1 complex that are proposed to contribute to DNA damage recognition.

Structural insights into the interaction of the crenarchaeal chromatin protein Cren7 with DNA

Cren7, a newly found chromatin protein, is highly conserved in the Crenarchaeota. The protein shows higher affinity for double‐stranded DNA than for single‐stranded DNA, constrains negative DNA supercoils in vitro and is associated with genomic DNA in vivo. Here we report the crystal structures of the Cren7 protein from Sulfolobus solfataricus in complex with two DNA sequences. Cren7 binds in the minor groove of DNA and causes a single‐step sharp kink in DNA (∼53°) through the intercalation of the hydrophobic side chain of Leu28. Loop β3‐β4 of Cren7 undergoes a significant conformational change upon binding of the protein to DNA, suggesting its critical role in the stabilization of the protein–DNA complex. The roles of DNA‐contacting amino acid residues in stabilizing the Cren7–DNA interaction were examined by mutational analysis. Structural comparison of Cren7‐DNA complexes with Sac7d‐DNA complexes reveals significant differences between the two proteins in DNA binding surface, suggesting that Cren7 and Sul7d serve distinct functions in chromosomal organization.

The mechanism of substrate-controlled allosteric regulation of SAMHD1 activated by GTP.

SAMHD1 is the only known eukaryotic deoxynucleoside triphosphate triphosphohydrolase (dNTPase) and is a major regulator of intracellular dNTP pools. It has been reported to be a potent inhibitor of retroviruses such as HIV-1 and endogenous retrotransposons. Previous crystal structures have revealed that SAMHD1 is activated by dGTP-dependent tetramer formation. However, recent data have indicated that the primary activator of SAMHD1 is GTP, not dGTP. Therefore, how its dNTPase activity is regulated needs to be further clarified. Here, five crystal structures of the catalytic core of SAMHD1 in complex with different combinations of GTP and dNTPs are reported, including a GTP-bound dimer and four GTP/dNTP-bound tetramers. The data show that human SAMHD1 contains two unique activator-binding sites in the allosteric pocket. The primary activator GTP binds to one site and the substrate dNTP (dATP, dCTP, dUTP or dTTP) occupies the other. Consequently, both GTP and dNTP are required for tetramer activation of the enzyme. In the absence of substrate binding, SAMHD1 adopts an inactive dimer conformation even when complexed with GTP. Furthermore, SAMHD1 activation is regulated by the concentration of dNTP. Thus, the level of dNTP pools is elegantly regulated by the self-sensing ability of SAMHD1 through a novel activation mechanism.

Structural and functional insights into lipid-bound nerve growth factors

Nerve growth factor (NGF) is a dimeric molecule that modulates the survival, proliferation, and differentiation of nervous cells and is also known to act on cells of the immune system and endocrine system. NGFs extracted from mouse submaxillary gland and cobra venom have different immunological behaviors, yet the underlying mechanism remains unclear. Here we report the crystal structure of the NGF purified from Chinese cobra Naja naja atra (cNGF), which unexpectedly reveals a 2‐tailed lipid molecule that is embedded between the two protomers of the NGF homodimer. In addition, crystallographic analysis indicated that the purified mouse NGF(mNGF) is free from lipid but can bind lysophosphatidylserine (lyso‐PS) in the same pocket as cNGF. Bioassays indicated that the binding of lipid molecules to cNGF and mNGF are essential for their mast cell activation activity and abates their p75NTR binding capacity. Taken together, these results suggest a new mechanism for the regulation of the function of NGF.—Tong, Q., Wang, F., Zhou, H.‐Z., Sun, H.‐L., Song, H., Shu, Y.‐Y., Gong, Y., Zhang, W.‐T., Cai, T.‐X., Yang, F.‐Q., Tang, J., Jiang, T. Structural and functional insights into lipid‐bound nerve growth factors. FASEB J. 26, 3811–3821 (2012). www.fasebj.org

Crystal structures of aprataxin ortholog Hnt3 reveal the mechanism for reversal of 5′-adenylated DNA

Aprataxin is a DNA deadenylase that resolves DNA 5′-AMP termini and reverses abortive DNA ligation. The crystal structures of Schizosaccharomyces pombe aprataxin Hnt3 in its apo form and in complex to dsDNA and dsDNA–AMP reveal how Hnt3 recognizes and processes 5′-adenylated DNA in a structure-specific manner. The bound DNA adopts a 5′-flap conformation that facilitates 5′-AMP access to the active site, where AMP cleavage occurs by a canonical catalytic mechanism.

Structural insights into the mechanism of calmodulin binding to death receptors

The death receptors Fas, p75(NTR) and DR6 are key components of extrinsically activated apoptosis. Characterization of how they interact with the adaptors is crucial in order to unravel the signalling mechanisms. However, the exact conformation that their intracellular death domain adopts upon binding downstream partners remains unclear. One model suggests that it adopts a typical compact fold, whilst a second model proposed an open conformation. Calmodulin (CaM), a major calcium sensor, has previously been reported to be one of the Fas adaptors that modulate apoptosis. This work reports that CaM also binds directly to the death domains of p75(NTR) and DR6, indicating that it serves as a common modulator of the death receptors. Two crystal structures of CaM in complexes with the corresponding binding regions of Fas and p75(NTR) are also reported. Interestingly, the precise CaM-binding sites were mapped to different regions: helix 1 in Fas and helix 5 in p75(NTR) and DR6. A novel 1-11 motif for CaM binding was observed in p75(NTR). Modelling the complexes of CaM with full-length receptors reveals that the opening of the death domains would be essential in order to expose their binding sites for CaM. These results may facilitate understanding of the diverse functional repertoire of death receptors and CaM and provide further insights necessary for the design of potential therapeutic peptide agents.

Insights into the interaction between Cren7 and DNA: the role of loop beta 3-beta 4

Sulfolobus synthesizes large amounts of small chromatin proteins Cren7 and Sul7d. The two proteins share overall structural similarity, but differ distinctly in the DNA-binding region between β3- and β4-strands. While Sul7d possesses a hinge of two amino acid residues, Cren7 contains a flexible seven-residue loop (loop β3–β4) in the region. Here, we report the role of loop β3–β4 in the interaction of Cren7 with duplex DNA. We show that all residues with a large side chain on the loop, i.e., Pro30, Lys31, Arg33 and Lys34, contributed significantly to the binding of Cren7 to DNA. The three basic amino acids affected the ability of Cren7 to constrain negative DNA supercoils in a residue number-dependent manner. The crystal structure of a complex between a mutant Cren7 protein (GR) with loop β3–β4 replaced by two residues (Gly and Arg) to mimic the hinge at the corresponding position in Sul7d and an 8-bp dsDNA has been determined. Structural comparison between the GR–DNA and Cren7–DNA complexes shows that GR resembles Sul7d more than Cren7 in DNA-binding size and in the effect on the width of the major groove of DNA and the pattern of DNA bending. However, GR induces smaller DNA curvature than Sul7d. Our results suggest that Cren7 and Sul7d package chromosomal DNA in a slightly different fashion, presumably permitting different chromosomal accessibility by proteins functioning in DNA transactions.

Roles of Leu28 side chain intercalation in the interaction between Cren7 and DNA

Crenarchaeal chromatin protein Cren7 binds double-stranded DNA in the minor groove, introducing a sharp single-step DNA kink. The side chain of Leu28, a residue conserved among all Cren7 homologs, intercalates into the kinked DNA step. In the present study, we replaced Leu28 with a residue containing a hydrophobic side chain of different sizes (i.e. L28A, L28V, L28I, L28M and L28F). Both the stability of the Cren7-DNA complex and the ability of Cren7 to constrain DNA supercoils correlated well with the size of the intercalated side chain. Structural analysis shows that L28A induces a kink (∼43°), nearly as sharp as that produced by wild-type Cren7 (∼48°), in the bound DNA fragment despite the lack of side chain intercalation. In another duplex DNA fragment, L28F inserts a large hydrophobic side chain deep into the DNA step, but introduces a smaller kink (∼39°) than that formed by the wild-type protein (∼50°). Mutation of Leu28 into methionine yields two protein conformers differing in loop β3-β4 orientation, DNA-binding surface and DNA geometry in the protein-DNA structure. Our results indicate that side chain intercalation is not directly responsible for DNA kinking or bending by Cren7, but plays a critical role in the stabilization of the Cren7-DNA complex. In addition, the flexibility of loop β3-β4 in Cren7, as revealed in the crystal structure of L28M-DNA, may serve a role in the modulation of chromosomal organization and function in the cell.