Structural studies on MRG701 chromodomain reveal a novel dimerization interface of MRG proteins in green plants

MRG proteins are conserved during evolution in fungi, flies, mammals and plants, and they can exhibit diversified functions. The animal MRGs were found to form various complexes to activate gene expression. Plant MRG1/2 and MRG702 were reported to be involved in the regulation of flowering time via binding to H3K36me3-marked flowering genes. Herein, we determined the crystal structure of MRG701 chromodomain (MRG701CD). MRG701CD forms a novel dimerization fold both in crystal and in solution. Moreover, we found that the dimerization of MRG chromodomains is conserved in green plants. Our findings may provide new insights into the mechanism of MRGs in regulation of gene expression in green plants.     

Molecular Determinants of PAM2 Recognition by the MLLE Domain of Poly(A)-Binding Protein

MLLE (previously known as PABC) is a peptide-binding domain that is found in poly(A)-binding protein (PABP) and EDD (E3 isolated by differential display), a HECT E3 ubiquitin ligase also known as HYD (hyperplastic discs tumor suppressor) or UBR5. The MLLE domain from PABP recruits various regulatory proteins and translation factors to poly(A) mRNAs through binding of a conserved 12 amino acid peptide motif called PAM2 (for PABP-interacting motif 2). Here, we determined crystal structures of the MLLE domain from PABP alone and in complex with PAM2 peptides from PABP-interacting protein 2. The structures provide a detailed view of hydrophobic determinants of the MLLE binding coded by PAM2 positions 3, 5, 7, 10, and 12 and reveal novel intermolecular polar contacts. In particular, the side chain of the invariant MLLE residue K580 forms hydrogen bonds with the backbone of PAM2 residues 5 and 7. The structures also show that peptide residues outside of the conserved PAM2 motif contribute to binding. Altogether, the structures provide a significant advance in understanding the molecular basis for the binding of PABP by PAM2-containing proteins involved in translational control, mRNA deadenylation, and other cellular processes.     

Functional elements on SIRPalpha IgV domain mediate cell surface binding to CD47

SIRPalpha and SIRPbeta1, the two major isoforms of the signal regulatory protein (SIRP) family, are co-expressed in human leukocytes but mediate distinct extracellular binding interactions and divergent cell signaling responses. Previous studies have demonstrated that binding of SIRPalpha with CD47, another important cell surface molecule, through the extracellular IgV domain regulates important leukocyte functions including macrophage recognition, leukocyte adhesion and transmigration. Although SIRPbeta1 shares highly homologous extracellular IgV structure with SIRPalpha, it does not bind to CD47. Here, we defined key amino acid residues exclusively expressing in the IgV domain of SIRPalpha, but not SIRPbeta1, which determine the extracellular binding interaction of SIRPalpha to CD47. These key residues include Gln67, a small hydrophobic amino acid (Ala or Val) at the 57th position and Met102. We found that Gln67 and Ala/Val57 are critical. Mutation of either of these residues abates SIRPalpha directly binding to CD47. Functional cell adhesion and leukocyte transmigration assays further demonstrated central roles of Gln67 and Ala/Val57 in SIRPalpha extracellular binding mediated cell interactions and cell migration. Another SIRPalpha-specific residue, Met102, appears to assist SIRPalpha IgV binding through Gln67 and Ala/Val57. An essential role of these amino acid residues in SIRPalpha binding to CD47 was further confirmed by introducing these residues into the SIRPbeta1 IgV domain, which dramatically converts SIRPbeta1 into a CD47-binding molecule. Our results thus revealed the molecular basis by which SIRPalpha binds to CD47 and shed new light into the structural mechanisms of SIRP isoform mediated distinctive extracellular interactions and cellular responses.     

The MLLE Domain of the Ubiquitin Ligase UBR5 Binds to Its Catalytic Domain to Regulate Substrate Binding

E3 ubiquitin ligases catalyze the transfer of ubiquitin from an E2-conjugating enzyme to a substrate. UBR5, homologous to the E6AP C terminus (HECT)-type E3 ligase, mediates the ubiquitination of proteins involved in translation regulation, DNA damage response, and gluconeogenesis. In addition, UBR5 functions in a ligase-independent manner by prompting protein/protein interactions without ubiquitination of the binding partner. Despite recent functional studies, the mechanisms involved in substrate recognition and selective ubiquitination of its binding partners remain elusive. The C terminus of UBR5 harbors the HECT catalytic domain and an adjacent MLLE domain. MLLE domains mediate protein/protein interactions through the binding of a conserved peptide motif, termed PAM2. Here, we characterize the binding properties of the UBR5 MLLE domain to PAM2 peptides from Paip1 and GW182. The crystal structure with a Paip1 PAM2 peptide reveals the network of hydrophobic and ionic interactions that drive binding. In addition, we identify a novel interaction of the MLLE domain with the adjacent HECT domain mediated by a PAM2-like sequence. Our results confirm the role of the MLLE domain of UBR5 in substrate recruitment and suggest a potential role in regulating UBR5 ligase activity.     

Molecular basis of the interaction of Saccharomyces cerevisiae Eaf3 chromo domain with methylated H3K36

Eaf3 is a component of both NuA4 histone acetyltransferase and Rpd3S histone deacetylase complexes in Saccharomyces cerevisiae. It is involved in the regulation of the global pattern of histone acetylation that distinguishes promoters from coding regions. Eaf3 contains a chromo domain at the N terminus that can bind to methylated Lys-36 of histone H3 (H3K36). We report here the crystal structures of the Eaf3 chromo domain in two truncation forms. Unlike the typical HP1 and Polycomb chromo domains, which contain a large groove to bind the modified histone tail, the Eaf3 chromo domain assumes an autoinhibited chromo barrel domain similar to the human MRG15 chromo domain. Compared with other chromo domains, the Eaf3 chromo domain contains a unique 38-residue insertion that folds into two short beta-strands and a long flexible loop to flank the beta-barrel core. Both isothermal titration calorimetry and surface plasmon resonance studies indicate that the interaction between the Eaf3 chromo domain and the trimethylated H3K36 peptide is relatively weak, with a K(D) of approximately 10(-4) m. NMR titration studies demonstrate that the methylated H3K36 peptide is bound to the cleft formed by the C-terminal alpha-helix and the beta-barrel core. Site-directed mutagenesis study and in vitro binding assay results show that the conserved aromatic residues Tyr-23, Tyr-81, Trp-84, and Trp-88, which form a hydrophobic pocket at one end of the beta-barrel, are essential for the binding of the methylated H3K36. These results reveal the molecular mechanism of the recognition and binding of the methylated H3K36 by Eaf3 and provide new insights into the functional roles of the Eaf3 chromo domain.     

Structural and functional characterization of the N-terminal domain of human Rad51D

Rad51D, one of five Rad51 paralogs, is required for homologous recombination and disruption of Holliday junctions with bloom syndrome protein (BLM) in vertebrates. The N-terminal domain of Rad51D is highly conserved in eukaryotic Rad51D orthologs and is essential for protein-protein interaction with XRCC2, but nothing is known about its individual structure or function. In this study, we determined the solution structure of the human Rad51D N-terminal domain (residues 1-83), which consists of four short helices flanked by long N- and C-terminal tails. Interestingly, the position of the N-terminal tail (residues 1-13) is fixed within the domain structure via several hydrophobic interactions between Leu4 and Thr27, Leu4 and Val28, and Val6 and Ile17. We show that the domain preferentially binds to ssDNA versus dsDNA and does not bind to a mobile Holliday junction by electrophoretic mobility shift assay. NMR titration and dynamics studies showed that human Rad51D-N interacts with ssDNA by positively charged and hydrophobic residues on its surface. The results suggest that the N-terminal domain of Rad51D is required for the ssDNA-specific binding function of human Rad51D and that the conserved N-terminal domains of other Rad51 paralogs may have distinguishable functions from each other in homologous recombination.