Structures of the L27 Domain of Disc Large Homologue 1 Protein Illustrate a Self-Assembly Module

Disc large 1 (Dlg1) proteins, members of the MAGUK protein family, are linked to cell polarity via their participation in multiprotein assemblies. At their N-termini, Dlg1 proteins contain a L27 domain. Typically, the L27 domains participate in the formation of obligate hetero-oligomers with the L27 domains from their cognate partners. Among the MAGUKs, Dlg1 proteins exist as homo-oligomers, and the oligomerization is solely dependent on the L27 domain. Here we provide biochemical and structural evidence of homodimerization via the L27 domain of Dlg1 from <i>Drosophila melanogaster</i>. The structure reveals that the core of the dimer is formed by a distinctive six-helix assembly, involving all three conserved helices from each subunit (monomer). The homodimer interface is extended by the C-terminal tail of the L27 domain of Dlg1, which forms a two-stranded antiparallel β-sheet. The structure reconciles and provides a structural context for a large body of available mutational data. From our analyses, we conclude that the observed L27 homodimerization is most likely a feature unique to the Dlg1 orthologs within the MAGUK family

Mechanism and Structure of γ‑Resorcylate Decarboxylase

γ-Resorcylate decarboxylase (γ-RSD) has evolved to catalyze the reversible decarboxylation of 2,6-dihydroxybenzoate to resorcinol in a nonoxidative fashion. This enzyme is of significant interest because of its potential for the production of γ-resorcylate and other benzoic acid derivatives under environmentally sustainable conditions. Kinetic constants for the decarboxylation of 2,6-dihydroxybenzoate catalyzed by γ-RSD from <i>Polaromonas</i> sp. JS666 are reported, and the enzyme is shown to be active with 2,3-dihydroxybenzoate, 2,4,6-trihydroxybenzoate, and 2,6-dihydroxy-4-methylbenzoate. The three-dimensional structure of γ-RSD with the inhibitor 2-nitroresorcinol (2-NR) bound in the active site is reported. 2-NR is directly ligated to a Mn²⁺ bound in the active site, and the nitro substituent of the inhibitor is tilted significantly from the plane of the phenyl ring. The inhibitor exhibits a binding mode different from that of the substrate bound in the previously determined structure of γ-RSD from <i>Rhizobium</i> sp. MTP-10005. On the basis of the crystal structure of the enzyme from <i>Polaromonas</i> sp. JS666, complementary density functional calculations were performed to investigate the reaction mechanism. In the proposed reaction mechanism, γ-RSD binds 2,6-dihydroxybenzoate by direct coordination of the active site manganese ion to the carboxylate anion of the substrate and one of the adjacent phenolic oxygens. The enzyme subsequently catalyzes the transfer of a proton to C1 of γ-resorcylate prior to the actual decarboxylation step. The reaction mechanism proposed previously, based on the structure of γ-RSD from <i>Rhizobium</i> sp. MTP-10005, is shown to be associated with high energies and thus less likely to be correct

Structural Insights into Thioether Bond Formation in the Biosynthesis of Sactipeptides

Sactipeptides are ribosomally synthesized peptides that contain a characteristic thioether bridge (sactionine bond) that is installed posttranslationally and is absolutely required for their antibiotic activity. Sactipeptide biosynthesis requires a unique family of radical SAM enzymes, which contain multiple [4Fe-4S] clusters, to form the requisite thioether bridge between a cysteine and the α-carbon of an opposing amino acid through radical-based chemistry. Here we present the structure of the sactionine bond-forming enzyme CteB, from <i>Clostridium thermocellum ATCC 27405</i>, with both SAM and an N-terminal fragment of its peptidyl-substrate at 2.04 Å resolution. CteB has the (β/α)₆-TIM barrel fold that is characteristic of radical SAM enzymes, as well as a C-terminal SPASM domain that contains two auxiliary [4Fe-4S] clusters. Importantly, one [4Fe-4S] cluster in the SPASM domain exhibits an open coordination site in absence of peptide substrate, which is coordinated by a peptidyl-cysteine residue in the bound state. The crystal structure of CteB also reveals an accessory N-terminal domain that has high structural similarity to a recently discovered motif present in several enzymes that act on ribosomally synthesized and post-translationally modified peptides (RiPPs), known as a RiPP precursor peptide recognition element (RRE). This crystal structure is the first of a sactionine bond forming enzyme and sheds light on structures and mechanisms of other members of this class such as AlbA or ThnB

The EZH2-SET domain C-terminus partially occupies the substrate binding groove.

<p>(a) The EZH2-SET (cyan) and hEHMT1-SET (orange) (PDB ID:3HNA) domains are superimposed and represented by ribbons. Zinc bound by hEHMT-SET is represented as a gray sphere. The substrate peptide bound by hEHMT1 is a yellow ribbon with the lysine side chain represented as sticks. The SAH bound by hEHMT1-SET is represented by sticks and colored by atom (carbon, yellow; oxygen, red; nitrogen, blue; sulfur, sienna). The C-terminal tail of EZH2-SET turns upwards and occupies the upper region of the substrate binding groove (red arrow pointing up). The C-terminus of hEHMT1-SET turns downward (red arrow pointing downward) forming the lower lobe of the cofactor binding pocket and coordinating one zinc atom. (b) The EZH2-SET (cyan) and SUV39H2 SET domain (magenta) (PDB ID:2R3A) crystal structures are superimposed and represented by ribbons. The C-termini in both structures occupy the collapsed substrate binding groove.</p

Additional disease-associated mutations outside this active site.

<p>The crystal structure of the EZH2-SET domain is represented as a ribbon model (cyan) with the hypothetical positions of cofactor and substrate (sticks colored by atom: carbon, yellow; oxygen, red; nitrogen, blue) extracted from the superimposed structure of EHMT1/PEPTIDE/SAH (PDB ID: 3HNA). EZH2-SET amino acid side chains are represented as sticks colored by atom (carbon, cyan; oxygen, red; nitrogen, blue). Secondary structure elements are labeled. (<b>a</b>) A V626M mutation was identified in WS [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084147#B21" target="_blank">21</a>]. This residue is located in the loop connecting β-1 and β-2 and may indirectly affect cofactor binding. (<b>b</b>) A K639E mutation was identified in WS [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084147#B21" target="_blank">21</a>]. This residue is located in the loop connecting β-2 and β-3. (<b>c</b>) R684 mutations were identified in WS (R>C) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084147#B21" target="_blank">21</a>], ETP ALL (R>H) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084147#B26" target="_blank">26</a>], and MF (R>C) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084147#B31" target="_blank">31</a>]. This residue does not participate in cofactor or substrate binding; however, its side chain does pack against α-4 which does participate in substrate binding in homologous SET domains. R690 mutations were identified in CMML (R>H) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084147#B17" target="_blank">17</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084147#B18" target="_blank">18</a>] and MDS (R>C) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084147#B28" target="_blank">28</a>]. This residue packs against F670 which in homologous SET domains contributes to the substrate lysine binding channel.</p

Structural context of Y646 and A682 mutations.

<p>The crystal structure of the EZH2-SET domain is represented as a ribbon model (cyan). Side chains are represented as sticks colored by atom (carbon, cyan; oxygen, red). Secondary structure elements are labeled. Y646 is completely buried in a hydrophobic cluster except for the solvent exposed tip of the phenyl ring where the phenyl oxygen forms a hydrogen bond with a water molecule. A682 is packed against the Y646 side chain distal to the catalytic site. Mutation of A682 likely indirectly affects substrate specificity by influencing the conformation of Y646 in the active state. Y646 and A682 mutations have been found in lymphoma [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084147#B24" target="_blank">24</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084147#B27" target="_blank">27</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084147#B33" target="_blank">33</a>], WS [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084147#B21" target="_blank">21</a>], and AML [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084147#B29" target="_blank">29</a>].</p

EZH2-SET mutations that may affect cofactor binding.

<p>The crystal structure of the EZH2-SET domain is represented as a ribbon model (cyan) with the hypothetical positions of cofactor and substrate (sticks colored by atom: carbon, yellow; oxygen, red; nitrogen, blue) extracted from the superimposed structure of EHMT1/PEPTIDE/SAH (PDB ID: 3HNA). EZH2-SET amino acid side chains are represented as sticks colored by atom (C, cyan; O, red; N, nitrogen). Secondary structure elements are labeled. Mutations at positions A692 {(DLBCL) (A>V) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084147#B23" target="_blank">23</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084147#B33" target="_blank">33</a>]}, N693 {(AMML) (N>T) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084147#B18" target="_blank">18</a>]; (ETP ALL) (N>Y) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084147#B26" target="_blank">26</a>]; (MF) (N>Y) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084147#B31" target="_blank">31</a>]}, and H694 {(WS) (H>Y) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084147#B22" target="_blank">22</a>]; (CMML) (H>R) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084147#B18" target="_blank">18</a>]} have been found in association with numerous diseases. All three mutations likely affect cofactor binding. An S695L mutation was identified in both WS [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084147#B21" target="_blank">21</a>] and ETP ALL [26]. This mutation may affect cofactor and substrate binding indirectly by influencing the conformation of residues in direct contact with these ligands. </p

Location of mutation in the second zinc binding domain of EZH2-SET.

<p>EZH2-SET (cyan) is represented as a ribbon diagram with zinc atoms shown as gray spheres and side chain represented as sticks (carbon, cyan; nitrogen, blue; sulfur, sienna) A C571Y mutation was identified in MF [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084147#B31" target="_blank">31</a>] and a C576W mutation was identified in MDS [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084147#B20" target="_blank">20</a>]. These mutations disrupt coordination of zinc in the second zinc binding domain likely destabilizing the protein. Additionally, a P577L mutation was observed in ETP ALL [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084147#B26" target="_blank">26</a>].</p

Location of mutation in the first zinc binding domain of EZH2-SET.

<p>EZH2-SET (cyan) is represented as a ribbon diagram with zinc atoms shown as gray spheres and side chain represented as sticks (carbon, cyan; nitrogen, blue; sulfur, sienna) A H530N mutation was identified in AML [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084147#B29" target="_blank">29</a>]. This mutation disrupts coordination of zinc in the first zinc binding domain likely having a strong destabilizing effect on the protein.</p

Mutations in the β-5/β-6 loop of EZH2-SET are contiguous with the putative substrate binding cleft.

<p>The crystal structure of the EZH2-SET domain is represented as a ribbon model (cyan). Side chains are represented as sticks colored by atom (carbon, cyan; oxygen, red; nitrogen, blue). Secondary structure elements are labeled. N673, L674, and N675 all interact directly with the C-terminal tail which occupies the substrate binding groove. Mutation of these residues could potentially affect substrate binding in the active state as well as the transition from the inactive to active state. An N673S mutation has been identified in CMML [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084147#B32" target="_blank">32</a>]. L674V mutations have been found in both MDS [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084147#B28" target="_blank">28</a>] and AML [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084147#B29" target="_blank">29</a>]. An N675K mutation was discovered in RCMD [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084147#B28" target="_blank">28</a>].</p