Solution structure of MSL2 CXC domain reveals an unusual Zn3Cys9 cluster and similarity to pre-SET domains of histone lysine methyltransferases.

The dosage compensation complex (DCC) binds to single X chromosomes in Drosophila males and increases the transcription level of X-linked genes by approximately twofold. Male-specific lethal 2 (MSL2) together with MSL1 mediates the initial recruitment of the DCC to high-affinity sites in the X chromosome. MSL2 contains a DNA-binding cysteine-rich CXC domain that is important for X targeting. In this study, we determined the solution structure of MSL2 CXC domain by NMR spectroscopy. We identified three zinc ions in the CXC domain and determined the metal-to-cysteine connectivities from (1)H-(113)Cd correlation experiments. The structure reveals an unusual zinc-cysteine cluster composed of three zinc ions coordinated by six terminal and three bridging cysteines. The CXC domain exhibits unexpected structural homology to pre-SET motifs of histone lysine methyltransferases, expanding the distribution and structural diversity of the CXC domain superfamily. Our findings provide novel structural insight into the evolution and function of CXC domains

Cryo-EM structure of an early precursor of large ribosomal subunit reveals a half-assembled intermediate

Abstract Assembly of eukaryotic ribosome is a complicated and dynamic process that involves a series of intermediates. It is unknown how the highly intertwined structure of 60S large ribosomal subunits is established. Here, we report the structure of an early nucleolar pre-60S ribosome determined by cryo-electron microscopy at 3.7 Å resolution, revealing a half-assembled subunit. Domains I, II and VI of 25S/5.8S rRNA pack tightly into a native-like substructure, but domains III, IV and V are not assembled. The structure contains 12 assembly factors and 19 ribosomal proteins, many of which are required for early processing of large subunit rRNA. The Brx1-Ebp2 complex would interfere with the assembly of domains IV and V. Rpf1, Mak16, Nsa1 and Rrp1 form a cluster that consolidates the joining of domains I and II. Our structure reveals a key intermediate on the path to establishing the global architecture of 60S subunits

NMR structure and dynamics of MSL2 CXC domain.

<p>(A) Structural superposition of the 20 lowest energy NMR structures. The Cα traces of residues 521–566 and three zinc ions (spheres) are shown in cross-eye stereoview. (B) Steady-state ¹H-¹⁵N heteronuclear NOE values are plotted as a function of residue number. The experiment was conducted for CXC-2, which contains residues 517–572 plus three extra N-terminal residues from the vector. No data were obtained for proline residues that lack amide proton. Error bars represent the experimental uncertainties estimated from the spectrum background noise.</p

Structural similarity between CXC and pre-SET domains.

<p>(A) Structure of MSL2 CXC domain. The Cys ligands and the C-terminal invariant Asn residue are shown as sticks, zinc ions as purple spheres and Zn-S coordination bonds and hydrogen bonds as dotted lines. (B) Structure of the SUV39 family Dim5 pre-SET domain (PDB code 1ML9). The long insertion between Cys-5 and Cys-6 is shown schematically as an oval. (C) Structure of the SET2 family NSD1 pre-SET domain (PDB code 3OOI). The three structures of MSL2 CXC, Dim5 pre-SET and NSD1 pre-SET domains are aligned by their Zn-Cys clusters. (D) The primary sequences of MSL2 CXC domain and pre-SET motifs of <i>Neurospora crassa</i> Dim5, human NSD1 and <i>Drosophila melanogaster</i> E(z). The observed or predicted zinc ligands are numbered sequentially from 1 to 9. The invariant C-terminal Asn is marked with asterisk. The coordination patterns of zinc ions observed in these structures are shown. The starting and ending residues of each sequence are labeled with residue numbers. A 48-residue region is omitted in DIM5 pre-SET. The zinc ligands in NSD1 pre-SET are numbered according to those in the CXC domain.</p

Refinement statistics for the 20 lowest energy NMR structures of MSL2 CXC domain.

<p>Refinement statistics for the 20 lowest energy NMR structures of MSL2 CXC domain.</p

The CXC domain of MSL2 binds three zinc ions.

<p>(A) ¹H-¹⁵N HSQC spectrum of the CXC domain. The spectrum was collected on 1 mM ¹⁵N-labeled CXC-3 protein in 50 mM phosphate buffer (pH 6.0) and 10% ²H₂O at 25°C and 600 MHz. The assigned residues are labeled with full-length MSL2 numbering, and the side-chain amide protons are connected by lines. (B) Electrospray ionization mass spectroscopy reveals three bound Zn²⁺ ions. The CXC-3 protein was exchanged into 200 mM ammonia acetate and analyzed with a Q-Star instrument. The inset shows the major isotopic peak series from a +5 charge species. The monoisotopic peak (m/z 1165.8361) corresponds to an exact mass of 5824.1805 Da, consistent with a complex of CXC-3 and three Zn²⁺ ions (monoisotopic MW = 5824.267 Da). The peak series around m/z = 1460 originates from a +4 charge species.</p