Crystal packing interactions of XOL-1 monomers. (A) Interface 1 consists of reciprocal interactions of regions 1 and 2 from neighboring monomers with a buried surface area of 2146 Ų per dimer. (B) Interface 2 consists of anti-parallel hydrogen bonding between β1-strands of neighboring monomers, creating a 12-stranded anti-parallel β-sheet (red) comprised of 6 strands from each monomer (β9, β10, β8, β11, β13, β1). (C) The central cavity of the tetramer in the crystal has a 45 Å diameter at its core. Secondary structure elements are colored as in Figure 3.

Comparison of XOL-1 and GHMP kinase structures. (A) The structure of XOL-1. Ribbon diagram of XOL-1 (PDB ID: 1MG7). Domain 1 consists of β-strands 2–7 and 12 (cyan) and α-helices 1–5 (yellow). Domain 2 consists of β-strands 8–11 (red) and α-helices 6–10 (green). Additional strands β1 and β13 and helix α1 form novel elements not found in known GHMP kinase structures. (B) The structure of homoserine kinase bound to ADP (PDB ID: 1FWK). Domain 1 β-strands and α-helices are cyan and yellow, respectively. Domain 2 β-strands and α-helices are red and green, respectively. ADP is represented by a ball and stick model. (C) The structure of mevalonate kinase (M. jannaschii; PDB ID: 1KKH). Colors are as in B.

XOL-1 ATP hydrolysis and binding. (A) In the presence of XOL-1 (10 μg; stippled bar), absorbance at 565 nm (Y-axis) is not significantly increased over the absorbance observed in any negative controls (hatched bars) in an enzyme-coupled colorimetric ATPase assay (see Materials and Methods). Negative controls (hatched bars) XOL-1, H-2K^b + 1 mM ATP, H-2K^b, Na⁺/K⁻-ATPase (5 milliunits), 1 mM ATP, buffer. As a positive control (gray bar), N⁺/K⁻-ATPase (5 milliunits) was incubated with 1 mM ATP. (B) The fluorescence emission of 20 mM ATP-TNP (○) is not significantly increased in the presence of 20 μM XOL-1 (▴). As a positive control, 20 μM hexokinase was incubated with 20 μM TNP-ATP (▵). Fluorescence emission intensity (Y-axis) is represented as function of wavelength (505–600 nm, X-axis). Excitation wavelength = 410 nm.

Genetic control of sex determination and dosage compensation in C. elegans. xol-1 is the primary sex-determination switch gene and the direct molecular target of the X-chromosome counting mechanism. (Top) Male (XO). High xol-1 activity specifies male fate by repressing the activity of sdc-1, sdc-2, and sdc-3 genes. Overt male sexual characteristics include a one-armed gonad (yellow) and a highly specialized tail used for mating. The dosage compensation machinery (colored ovals) does not bind to the X chromosome. Thus, X chromosome gene expression is unregulated (red chromosome). DC, dosage compensation complex (i.e., DPY-26, DPY-27, DPY-28, and MIX-1). (Bottom) Hermaphrodite (XX). Low xol-1 activity specifies hermaphrodite fate by allowing the activation of sdc-1, sdc-2, and sdc-3 genes. Overt hermaphrodite sexual characteristics include a two-armed gonad (yellow, with brown oocytes and embryos) and a tapered tail. The sdc-1,sdc-2, and sds-3 gene products (colored rectangles) recruit a large complex of dosage compensation proteins (DC, colored ovals) to both X chromosomes, repressing X gene expression by half (brown chromosome).

The putative active-site of XOL-1. (A) The cavity that represents the putative active-site of XOL-1 and, in other GHMP kinases, binds ATP is represented as an electrostatic surface nestled between domains 1 and 2. Red is electronegative and blue is electropositive (calculated in GRASP; Nicholls et al. 1991; −15 to +15 kT/e). Secondary structure elements are colored as in Figure 2A. Side chains of charged and polar residues lining the cavity are represented by ball and stick. (B) Same as A, but with view rotated by ∼180°. The location of the Cα carbon of Gly 123 is represented by a red dot. (C) The cavity that represents the putative active site of XOL-1 is represented as a surface colored by conservation. Side chains of residues identical between C. elegans and C. briggsae XOL-1 are in dark blue, whereas those conservatively substituted are in cyan with the corresponding colors mapped onto the surface. The strongly conserved region encompassing motif3 (β9–β10) is colored dark blue. (D) Same as C, but with view rotated by ∼180°.

Sequence alignment of XOL-1. (A) Structure-based sequence alignment of XOL-1 within GHMP kinase conserved motif regions. Sequence of XOL-1 (bottom) is aligned with structurally equivalent residues from other GHMP kinases. The three GHMP conserved motifs are highlighted in yellow. In XOL-1, these residues are as follows: motif1, residues P45-S53; motif2, residues D120-P128; and motif3, V298-G302. Tandem serines of motif2 are highlighted by boxes. The sequence of XOL-1 is highly divergent, even within conserved motifs, from other GHMP kinases with only one structurally equivalent residue found to be identical (Gly 123 in motif2) throughout the entire sequence. Residues conserved identically in all structures are in red. Highly conserved residues are in blue. Secondary structure is assigned above. PMK, phosphomevalonate kinase; MVK_mj, mevalonate kinase (M. jannaschii); MVK_rn, mevalonate kinase (R. norvegicus); MVPD, mevalonate-5-phosphate decarboxylase; HSK, homoserine kinase; XOL_c, XOL-1 (C. briggsae); XOL_ce, XOL-1 (C. elegans). (B) Alignment of C. elegans and C. briggsae XOL-1 sequences. The C. elegans and C. briggsae XOL-1 sequences were aligned by the clustal method (GONNET series). Gaps and insertions incompatible with the structure were edited manually. Residues corresponding to GHMP conserved motifs are highlighted in yellow. A conserved acidic C-terminal motif is highlighted in green. Identically conserved residues are highlighted in red and conservatively substituted residues are in blue. Secondary structure is assigned above.