Comparison of KH domain structures. (A) Backbone trace of αCP1-KH3 (grey) shown in stereo superimposed with those of other KH domain structures as listed. These include KH domain structures both in the absence and presence of bound oligonucleotide. (B) The α-carbon deviation for each KH domain residue from the corresponding aligned residue of αCP1-KH3 is plotted versus the αCP1-KH3 residue number. Amino acids >3.5 Å, or with no corresponding aligned residue, are indicated with an off-scale score (>5).

Interaction of the αCP1-KH3 with the 30 nt of the 3′-UTR of AR mRNA measured by surface plasmon resonance. (A) 30 RU RNA was immobilized on a streptavidin-coated chip. Binding interactions were measured for a series of dilutions of the αCP1-KH3 domain from 10 to 0.625 μM for 2 min using flow rate of 50 μl/min. (B) Steady-state analysis of the interaction yielded a K_d value of 4.37 μM.

The crystal structure of αCP1-KH3 (residues 279–356) solved to 2.1 Å resolution depicted in (A) cartoon form and (B) as a molecular surface in the same orientation. The structure is shown from the beginning of β-strand 1 to the end of α-helix 3, since the regions outside these bounds were random coil or not visible in the density. The GXXG motif, common to this oligonucleotide-binding motif, is coloured blue. The ‘variable loop’ region between β-sheets 2 and 3 is coloured pink. These regions bound the hydrophobic oligonucleotide-binding cleft that accommodates C-rich RNA or ssDNA. (C) The electrostatic potential emanating from the αCP1-KH3 structure calculated using the APBS software package () (39–43). Potential contours are shown at +1 kT/e (blue) and −1 kT/e (red) and obtained by solution of the linearized Poisson–Boltzmann equation at 150 mM ionic strength with a solute dielectric of 2 and a solvent dielectric of 78.5. The blue contour represents a striking positive potential directing oligonucleotides to the binding cleft.

Structure-based sequence alignment of seven KH domains of high structural similarity to αCP1-KH3. Each KH domain was structurally aligned using LSQMAN (36) against αCP1-KH3. Amino acid residues with α-carbon positions within 3.5 Å of a corresponding αCP1-KH3 residue are shown in black. Highlighted in purple are the amino acid residues that do not align well with residues of αCP1-KH3. Secondary structural elements, as defined in Lewis et al., (23) are shown above the corresponding sequence in cartoon form. Parenthesized numbers represent the amino acid numbers at the start and finish of the superimposed core region for each structure, and indicate the extent of the structure used to calculate sequence identity with αCP1-KH3 (final column). The GXXG motif and the variable loop regions are blocked with grey. Amino acid residues reported to make contact with the oligonucleotide [in the cases of structures determined in complex with either RNA or ssDNA (25–27)] are highlighted in red, and the αCP1-KH3 predicted to make contact with oligonucleotide in the current study are highlighted in tan. NMR structures were structurally aligned on the basis of the first chain in the deposited PDB coordinate file and all were deemed to be representative of the set of structures.

(A) Molecular surface of αCP1-KH3 showing modelled position of poly(C)-RNA (orange) based on the Nova2-KH3-RNA structure (accession no. 1EC6). The poly(C)-tetrad is viewed from above the GXXG and variable loops, highlighting their position either side of the hydrophobic binding cleft. (B) Summary of potential interactions occurring between the modelled αCP1-KH3 and poly(C)-RNA. A poly(C)-RNA-tetrad is represented schematically. Potential hydrogen bond interactions are indicated by dotted lines. Those from specific residue atoms to the RNA backbone are listed on the right, and those to the cytosine bases are listed on the left. The red dotted lines represent intra-molecular hydrogen bonds that may stabilize the RNA in its binding mode to the KH domain. Hydrophobic or van der Waals contacts to the cytosine bases are indicated by solid lines. (C) The positions of Arg 32 and Arg 51 side chains are highlighted beneath the molecular surface of αCP1-KH3. Potential hydrogen bonds to the poly(C)-RNA are shown as dotted lines.