Abstract
Drosophila melanogaster Heterochromatin Protein 1a (HP1a) is an essential protein critical for heterochromatin assembly and regulation. Its chromo shadow domain (CSD) homodimerizes, a requirement for binding protein partners that contain a PXVXL motif. How does HP1a select among its many different PXVXL-containing partners? HP1a binds tightly to Heterochromatin Protein 2 (HP2), but weakly to PIWI. We investigated differences in homodimerization and the impact of the C-terminal extension (CTE) by contrasting HP1a to its paralogue, HP1b. HP1a and HP1b differ in the dimerization interface, with HP1a having an Arg at position 188 rather than Glu. We find that while this substitution reduces the dimerization constant, it does not impact the binding surface as demonstrated by unchanged partner binding affinities. However, the CTE (only 4 residues in HP1a as compared with 87 residues in HP1b) is critical; the charged residues in HP1a are necessary for tight peptide binding. Examining a panel of amino acid substitutions in the HP1a CSD, we find that Leu-165 in HP1a interacts with HP2 but not PIWI, supporting the conclusion that different sites in the binding surface provide discrimination for partner selection. Partner sequence is also critical for affinity, as the remaining difference in binding between HP2 and PIWI polypeptides is eliminated by swapping the PXVXL motifs between the two. Taken together, these studies indicate that the binding surface of the HP1a CSD plus its short CTE provide the needed discrimination among HP1a's partners, and that the CTE is important for differentiating the interactions of the Drosophila HP1 paralogs.
Highlights
HP1a (Heterochromatin Protein 1a) discriminates among its partners, binding Heterochromatin Protein 2 (HP2) 66-fold more tightly than PIWI
Examining a panel of amino acid substitutions in the HP1a chromo shadow domain (CSD), we find that Leu-165 in HP1a interacts with HP2 but not PIWI, supporting the conclusion that different sites in the binding surface provide discrimination for partner selection
The D. melanogaster Dimerization Interface of HP1a Is Weaker Than That of Several Homologues—We previously determined the crystal structure of the HP1a chromo shadow domain, and found that the dimerization interface composed of nine residues (Fig. 1A) [12]
Summary
HP1a (Heterochromatin Protein 1a) discriminates among its partners, binding HP2 66-fold more tightly than PIWI. Partner sequence is critical for affinity, as the remaining difference in binding between HP2 and PIWI polypeptides is eliminated by swapping the PXVXL motifs between the two Taken together, these studies indicate that the binding surface of the HP1a CSD plus its short CTE provide the needed discrimination among HP1a’s partners, and that the CTE is important for differentiating the interactions of the Drosophila HP1 paralogs. To begin to understand some of the features of HP1a that give it its unique distribution and partner interactions, we have 1) analyzed its dimerization properties compared with those of HP1b, and 2) used mutagenesis to determine the amino acids responsible for tight HP2 binding, in contrast to weak PIWI binding, to the chromo shadow domain. Our results demonstrate that the amino acid sequence of the binding partner together with the HP1a binding surface determines the strength of interaction
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