Malignant brain tumor repeats: a three-leaved propeller architecture with ligand/peptide binding pockets

Abstract

We report on the X-ray structure of three 100-amino acid mbt repeats in h-l(3)mbt, a polycomb group protein involved in transcriptional repression, whose gene is located in a region of chromosome 20 associated with hematopoietic malignancies. Interdigitation between the extended arms and cores of the mbt repeats results in a three-leaved propeller-like architecture, containing a central cavity. We have identified one ligand binding pocket per mbt repeat, which accommodates either the morphilino ring of MES or the proline ring of the C-terminal peptide segment, within a cavity lined by aromatic amino acids. Strikingly, phenotypic alterations resulting from point mutations or deletions in the mbt repeats of the related Drosophila SCM protein are clustered in and around the ligand binding pocket.

Figure 1. Sequence Alignments of Proteins Containing mbt Repeats

(A) Multiple sequence alignment of the N-terminal arms of 3mbt repeats from h-l(3)mbt and related family members using CLUSTALW. Secondary structure elements defined by crystal structure of 3mbt repeats are shown above the alignment. α helices, wavy magenta lines; 3₁₀ helices, wavy orange lines; β strands, arrows; loops, straight lines. The helices are numbered consecutively from the N terminus. Helical structure αA, featured with a dashed line, only exists in the second and third repeats, while αD exists only in the first repeat. The loop with a dashed line only exists in the second and third repeats. Conserved hydrophobic, aromatic, positively charged, negatively charged, and polar residues are marked with yellow, green, red, blue, and orange. Accession numbers (SWISS-PORT, Genbank, or PIR) are as follows: H. sapiens (brain) l(3)mbt, Q9Y468; H. sapiens (brain) KIAA1798, Q96JM7; H. sapiens (testis), Q9Y4Q9; D. melanogaster, Q9VB52; M. fascicularis, Q95LL9; C. elegans (R06c7.7), Q21769; C. elegans (Y48GIA.6), Q9N3Q7; D. melanogaster SCM, Q9VHA0. (B) Multiple sequence alignment of the β subunit core of three mbt repeats from h-l(3)mbt and related family members, with defined secondary structure elements as indicated in (A). The β2 region featured with a dashed line only exists in first repeat, while β6 only exists in the first and second repeats. The loop featured with a dashed line only exists in third repeat. Structure-based sequence alignment of the Tudor domain with the mbt β subunit core is shown at the bottom. Mutated residues in SCM and residues of the Tudor domain involved in binding Sm proteins and the GAR domain are boxed. Conserved residues that form the putative drug/peptide binding pocket are highlighted with asterisks, those involved in hydrogen bond formation (Figure 3D) are indicated by closed circles, and those involved in the hydrophobic pentad motif (Figure 3E) are indicated by open circles. The accession number for H. sapiens SMN1-Tudor is Q16637.

Figure 2. 3mbt Adopts a Triangular Propeller-like Architecture

(A) Ribbon representation (orthorhombic crystal) of the structure (β subunit core face) of 3mbt. The first (residues 204–309), second (residues 310–416), and third (residues 417–527) mbt repeats are colored cyan, red, and yellow, respectively. Secondary structure elements are indicated for the first mbt repeat, which comprises a long N-terminal arm (αB, αC, and αD) and a β subunit core (β1, β2, β3, β1, β4, β5, α2, and β6). αA exists only in the second and third repeats and is indicated explicitly for clarity. α1 of the second repeat is shown to clarify the unique kissing α1–β 2 interaction (see text and Figure 4C). (B) Electrostatic surface representation (orthorhombic crystal) of the β subunit core face of the triangular propeller-like 3mbt structure. Blue, electropositive; red, electronegative. This face, with its extensive acidic (red) patches, contains three ligand binding pockets (numbers 1, 2, and 3 refer to the pockets belonging to the first, second, and third repeats, respectively). (C) Electrostatic surface representation (orthorhombic crystal) of the opposite bowl-like face of 3mbt. This face, which contains basic (blue) patches on the periphery, is comprised of N-terminal arms and predominantly exhibits nonpolar character. Some electropositive charge is clustered toward one side of the surface. A central cavity with approximate dimensions of 10 Å × 15 Å can be clearly seen in both (B) and (C).

Figure 3. Long N-Terminal Arm and β Subunit Core Motifs of 3mbt

(A) Side view of the 3mbt structure (orthorhombic crystal) reveals that the N-terminal arms and β subunit cores are located along different faces of the molecule. The N-terminal arms of each repeat (colored as in Figure 2A) are located along one face (bottom), exhibiting a bowl-like feature, while the β subunit cores (silver) are located on the opposite face (top), being positioned on top of the bowl. The structurally conserved element αA-linker-βB forms a handle-like feature along three sides of the molecule. (B) Superimpositions of the first (cyan), second (red), and third (yellow) repeats of 3mbt (orthorhombic crystal). The β subunit cores superposition well on each other (boxed region), with resulting rmsd’s of 1.0 Å for the Cα atoms. By contrast the attached N-terminal arms are oriented in different directions. (C) Superimpositions of the N-terminal arms from the first (cyan), second (red), and third (yellow) repeats of 3mbt (orthorhombic crystal). The αB-linker-αC segments superposition well on each other (boxed region; rmsd’s of 0.9 for Cα atoms), while the C-terminal segments are oriented in different directions. An element of 3₁₀-helical turn, αC, is only observed for the arm of the first repeat. (D) Ribbon drawing showing interdomain hydrogen bonding contacts between residues from the αA-linker-αB segment of the first N-terminal arm (cyan) and the β subunit core and N-terminal arm from the third mbt repeat (yellow). Hydrogen bond networks are indicated by dashed magenta lines. (E) Ribbon drawing showing interdomain stacking interactions between aromatic and proline residues from the first N-terminal arm (cyan) and the β subunit core and the N-terminal arm from the third repeat (yellow).

Figure 4. Details of Interdomain Interactions in Orthorhombic 3mbt Structure

(A) Worm representation of the superimpositions of all determined structures (seleno-met, native I, and native II) of 3mbt repeats (Table 1). The resulting average rmsd’s for all main chain atoms are 0.75 Å Regions that superposition well are indicated with thin worm representation, while regions that superposition less well are indicated with thicker worm representation. The most flexible regions are the handle-like segments of the second and third repeats. The 2F_o − F_c electron density map of the central cavity and interdomain interactions of 3mbt repeats at 1.55 Å resolution, contoured at 1.5 σ, with the refined atomic model in a ball and stick representation. Residues from first, second, and third mbt repeat are colored cyan, red, and yellow, as in Figure 2A. Oxygen, nitrogen, and sulfur atoms are colored red, cyan, and green, respectively. (B) View showing inter-β subunit core contacts from the first (cyan) and second (red) repeats. An additional interaction is mediated by a kissing α1–β2 region that involves D384 (β1 from second repeat) and S253 (β2 from first repeat). (C) View showing inter-β subunit core contacts from the first (cyan) and third (yellow) repeats. The interactions involve unique residues compared with (B). (D) View showing interdomain contacts mediated by a unique residue, R444, from the interdigitated region of the third N-terminal arm (yellow) that pairs with residue D391 from the second β subunit core (red) and residue D497 from the third β subunit core (yellow) via hydrogen bond networks (dotted magenta lines). (E) View focuses on the bound SO₄²⁻ molecule in the central cavity of the 3mbt triangular propeller structure. The SO₄²⁻ molecule is anchored in the central cavity via hydrogen bond networks (dotted green lines) with residues R460 and H498.

Figure 5. MES Ligand Binding Pockets in Trigonal 3mbt Structures

(A) Superimposition of three mbt repeats with bound MES molecules in the seleno-met structure. A MES molecule (labeled 1, 2, and 3) is bound to each β subunit core (colored as in Figure 2A). (B) The mutation sites are located in and around the MES binding pocket (second mbt repeat) in the 3mbt structure (seleno-met). The morphilino ring of the bound MES molecule (red) is encapsulated by polar and aromatic residues (silver). The electron density for bound MES is shown in a chicken wire representation. Residues that are point mutated (magenta) and deleted (blue) are highlighted with the letters M and D, respectively. Residue M357 in the second repeat is included, since it occupies a position analogous to R461 in the third repeat (see [C] and [D]). Hydrogen bond networks are indicated by dotted lines (magenta). Oxygen and nitrogen atoms are colored red and blue, respectively. (C) The surface representation of the open form of the MES (yellow) binding pocket from the third mbt repeat in the seleno-met structure. Residues lining this pocket are indicated in black. Arginine R461 is indicated in dotted surface (pink) representation with its side chain in a stick mode (white). Conserved residues among mbt repeats that are mutated in the SCM protein (Bornemann et al., 1998) are explicitly indicated in blue (deletion mutations, labeled D) and red (point mutations, labeled M). (D) The surface representation of the closed form of the MES binding pocket from the third mbt repeat in the GTP-containing native 1 structure. The R461 arginine finger flips its long and bulky side chain into the pocket, closing this site to MES ligands. This alignment is stabilized by a single hydrogen bond with residue D459 (point mutated in mbt of SCM protein; Bornemann et al., 1998).

Figure 6. C-Terminal Peptide Binding Pocket in Orthorhombic 3mbt Structure

(A) Packing of adjacent 3mbt molecules (cyan and gold) in the trigonal crystal, resulting in a total buried surface area of 314 Ų per monomer (B and C) Two views of packing of adjacent 3mbt molecules (cyan and gold) in the GTP-preincubated native II orthorhombic structure, resulting in a total buried surface area of 1663 Ų surface area per monomer. Note mutual insertion of C-terminal loops into ligand binding pockets of the first repeat (seen clearly in [C]). (D) The surface representation of the binding pocket from the first repeat of 3mbt, which accommodates the C-terminal loop from a symmetry related 3mbt molecule. The peptide backbone of the C-terminal loop is colored orange, and its side chains are indicated in yellow. The side chain of residue Q250, represented as a dotted pink surface, is directed away from the pocket, analogous to the R461 arginine finger in Figure 5C. Mutations sites and cage residues are colored as in Figure 5C. (E) The details of the first repeat pocket with the bound C-terminal loop. The residues involved in the binding pocket are shown in ball and stick representation (silver). Only residues 522–524 of the C-terminal segment are shown in the ball and stick representation in the interest of clarity, and the electron density map of this segment is shown in a chicken wire representation. Mutations sites and oxygen and nitrogen atoms are colored as in Figure 5B. The intermolecular hydrogen bond networks between C-terminal loop and cage residues are shown as dashed magenta lines. The main chain atoms of certain residues are shown explicitly in ball and stick representation for clarity.

Figure 7. Structural Homology between the 3mbt β Subunit Core, Tudor Domain, and Chromodomain

(A) Ribbon representation showing the β subunit core of the second repeat of the seleno-met 3mbt structure. (B) Ribbon representation of the Tudor domain (Selenko et al., 2001). Note the difference in the position of the α1 helical turn between Tudor domain (B) and mbt repeat (A). (C) Ribbon representation of the chromodomain of HP1 bound to a dimethylated lysine 9 histone H3 tail (Jacobs and Khorasanizadeh, 2002). (D) Electrostatic surface representation of the second mbt repeat of the seleno-met 3mbt structure. Refer to Figure 2B for the electrostatic surface of the ligand binding cage. (E) Electrostatic surface representation of the Tudor domain. Note the differences in surface charge distributions between the Tudor domain (E) and mbt repeat (D). (F) Electrostatic surface representation of the chromodomain of HP1. Note the cavity and the aromatic cage that accommodates the histone H3 peptide tail and dimethylated lysine 9. The secondary-structural elements of α1 and β4 (A and B) are colored magenta and cyan, respectively. Hydrophobic (yellow), positively charged (red), negatively charged (blue), and polar (orange) side chains are shown in the ball and stick representation for 3mbt in (A) and the Tudor domain in (B). Other related residues involved in the cage are indicated in silver for clarity. The electrostatic surface in 3mbt (D) and the Tudor domain (E) is colored as in Figures 2B and 2C. The red arrow indicates the location of the ligand binding cage in the mbt structure and the binding cavity for the histone H3 peptide tail in the chromodomain, respectively.