Detection and quantification of Cul3-CHIM11/12^BTB binding by gel filtration and ITC experiments.

<p>(A) Gel filtration elution profile of CHIM11/12^<b>BTB</b> after the mixing with Cul3^<b>NTD</b>. (B) ITC characterization of the interaction of CHIM11/12^<b>BTB</b> with Cul3^<b>NTD</b>. The insets report the SDS-PAGE analysis of the peaks.</p

MOESM1 of Purification of viable peripheral blood mononuclear cells for biobanking using a robotized liquid handling workstation

Additional file 1: Figure S1 Purification of mononuclear cells from a peripheral blood sample enriched with HL60 cells to gain a WBC cell density of 30 × 106 cells/mL. HL60 (blue), mononuclear cells (red) and granulocytes (green)were detectable according to the FSC-A vs SSC-A dot-plot. Granulocytes’ population is lost after PBMC automatic isolation, while the percentage of HL60 and mononuclear cells were enriched. CD45 vs CD33 dot-plot is shown to better identify the HL60 cells that derive from CD33 positive human acute myeloid leukemia (M2)

Percentages of sequence identity and number of aligned residues of the BTB domains of selected KCTD members are reported on the right and left side of the diagonal, respectively.

<p>In addition to the proteins here characterized (KCTD6, KCTD11, KCTD12, and KCTD15), we included into the comparison representative members (KCTD5, KCTD7, KCTD13, BTBD10 and SHKBP1) of KCTD subgroups whose interaction with Cul3 has been experimentally demonstrated.</p><p>Percentages of sequence identity and number of aligned residues of the BTB domains of selected KCTD members are reported on the right and left side of the diagonal, respectively.</p

Cullin 3 Recognition Is Not a Universal Property among KCTD Proteins

<div><p>Cullin 3 (Cul3) recognition by BTB domains is a key process in protein ubiquitination. Among Cul3 binders, a great attention is currently devoted to KCTD proteins, which are implicated in fundamental biological processes. On the basis of the high similarity of BTB domains of these proteins, it has been suggested that the ability to bind Cul3 could be a general property among all KCTDs. In order to gain new insights into KCTD functionality, we here evaluated and/or quantified the binding of Cul3 to the BTB of KCTD proteins, which are known to be involved either in cullin-independent (KCTD12 and KCTD15) or in cullin-mediated (KCTD6 and KCTD11) activities. Our data indicate that KCTD6^BTB and KCTD11^BTB bind Cul3 with high affinity forming stable complexes with 4:4 stoichiometries. Conversely, KCTD12^BTB and KCTD15^BTB do not interact with Cul3, despite the high level of sequence identity with the BTB domains of cullin binding KCTDs. Intriguingly, comparative sequence analyses indicate that the capability of KCTD proteins to recognize Cul3 has been lost more than once in distinct events along the evolution. Present findings also provide interesting clues on the structural determinants of Cul3-KCTD recognition. Indeed, the characterization of a chimeric variant of KCTD11 demonstrates that the swapping of α2β3 loop between KCTD11^BTB and KCTD12^BTB is sufficient to abolish the ability of KCTD11^BTB to bind Cul3. Finally, present findings, along with previous literature data, provide a virtually complete coverage of Cul3 binding ability of the members of the entire KCTD family.</p></div

Multiple sequence alignment of the BTB domains of different KCTD proteins (KCTD5, KCTD6, KCTD11, KCTD12, KCTD15).

<p>The sequence of the novel chimeric construct CHIM11/12BTB is also reported.</p

Root mean square fluctuations per residue of KCTD11^BTB and CHIM11/12^BTB.

<p>RMSF values calculated on C^α atoms in the equilibrated region the trajectories (20–100 ns) for the simulations carried out on KCTD11^<b>BTB</b> (A) and CHIM11/12^<b>BTB</b> (B). Secondary structure elements are represented as bars. Helices and strands are colored in blue and red, respectively. In the insets the RMSF values of the α2β3 loops, within the different amino acid sequences, are reported.</p

Detection of Cul3-KCTDs binding by gel filtration.

<p>Gel filtration elution profiles of KCTD6^<b>BTB</b> (A), KCTD11^<b>BTB</b> (B), KCTD12^<b>BTB</b> (C), and KCTD15^<b>BTB</b>(D) after their mixing with Cul3^<b>NTD</b>. The insets report the SDS-PAGE analysis of the peaks.</p

Biophysical characterization of KCTD15^BTB conducted by Far-UV CD spectroscopy (A) and by light scattering (B).

<p>The experiments were carried out in a 20mM sodium phosphate buffer (pH 7.5) containing 2 mM DTT.</p

Biophysical characterization of CHIM11/12^BTB conducted by Far-UV CD spectroscopy (A) and by light scattering (B).

<p>The dashed line in (A) represents the far-UV CD spectrum of KCTD11^<b>BTB</b>. The experiments were carried out in a 20mM sodium phosphate buffer (pH 7.5) containing 2 mM DTT.</p

Cul3 binding site in the average MD structures of KCTD11^BTB and CHIM11/12^BTB.

<p>KCTD11^<b>BTB</b>, CHIM11/12^<b>BTB</b>, Cul3^<b>NTD</b> are represented in blue, red and green, respectively. For clarity, a single chain of the tetramers is highlighted.</p