Table 2.

Elucidation of the binding surface of the HP1a-CSD. This table summarizes an exploration of some of the factors that contribute to HP1a-CSD ligand binding. Line 1. HP1a-CSD binds to the HP2 peptide (either wild-type or with a C2467S substitution) with tenths of micromolar affinity, tenfold less than the dimerization constant of HP1a-CSD. HP1a-CSD binds to the PIWI peptide with tens of micromolar affinity, tenfold more than the dimerization constant of HP1a-CSD. Lines 2–3. Consistent with the behavior of known PXVXL platform binding peptides, the HP1a W200A and I191E substitutions abrogate binding of the HP2 and PIWI peptides. Line 4. Although PXVXL is the published consensus polypeptide sequence for binding to the HP1a-CSD dimer, LXVXI can bind equally well in the context of PIWI (compare lines 1 and 4 in the PIWI Kd column). Line 5. Removing the last three residues of HP1a-CSD weakens peptide binding 3- to 16-fold, while not shifting the dimerization constant appreciably. Line 6. HP2 is impacted twice as much as PIWI is by mutating HP1a E195. Line 7. An equivalent fold change occurs when HP1a A174 is mutated. Line 8. The phosphorylation mimic mutations in HP1a-CSD decrease the dimerization constant of HP1a-CSD while improving the binding of the HP2 22mer and the PIWI 21mer.

HP1a CSD Construct	Peptide	HP1a Dimerization Constant	Kd for HP2 (μM)	Kd for PIWI (μM)
WT	HP2 (HP2 LCVKI) PIWI	3.0±0.2	0.30±0.02 (0.14±0.07)	20.0±0.5
I191E	HP2 PIWI	N/A	>400	>2000
W200A	HP2 PIWI	5.8±0.6	>500	>2000
WT	Pentamer Swap	3.0±0.2	0.99±0.08	21.3±1.3
Δ204	HP2 PIWI	2.0±0.2	4.9±0.6	60±2
E195Q	As above	4.3±0.4	3.7±0.4	97±12
A174E	As above	(3.0±0.2)	1.26±0.12	101±6
S199ES202E	As above	19±1	0.044±0.007	1.7±0.1