The eight mammalian Cbx proteins are chromodomain-containing proteins involved 
in regulation of heterochromatin, gene expression, and developmental programs. 
They are evolutionarily related to the Drosophila HP1 (dHP1) and Pc (dPc) 
proteins that are key components of chromatin-associated complexes capable of 
recognizing repressive marks such as trimethylated Lys-9 and Lys-27, 
respectively, on histone H3. However, the binding specificity and function of 
the human homologs, Cbx1-8, remain unclear. To this end we employed structural, 
biophysical, and mutagenic approaches to characterize the molecular determinants 
of sequence contextual methyllysine binding to human Cbx1-8 proteins. Although 
all three human HP1 homologs (Cbx1, -3, -5) replicate the structural and binding 
features of their dHP counterparts, the five Pc homologs (Cbx2, -4, -6, -7, -8) 
bind with lower affinity to H3K9me3 or H3K27me3 peptides and are unable to 
distinguish between these two marks. Additionally, peptide permutation arrays 
revealed a greater sequence tolerance within the Pc family and suggest 
alternative nonhistone sequences as potential binding targets for this class of 
chromodomains. Our structures explain the divergence of peptide binding 
selectivity in the Pc subfamily and highlight previously unrecognized features 
of the chromodomain that influence binding and specificity.