RESUMO
The self-association of sterile alpha motifs (SAMs) into a helical polymer architecture is a critical functional component of many different and diverse array of proteins. For the Drosophila Polycomb group (PcG) protein Polyhomeotic (Ph), its SAM polymerization serves as the structural foundation to cluster multiple PcG complexes, helping to maintain a silenced chromatin state. Ph SAM shares 64% sequence identity with its human ortholog, PHC3 SAM, and both SAMs polymerize. However, in the context of their larger protein regions, PHC3 SAM forms longer polymers compared with Ph SAM. Motivated to establish the precise structural basis for the differences, if any, between Ph and PHC3 SAM, we determined the crystal structure of the PHC3 SAM polymer. PHC3 SAM uses the same SAM-SAM interaction as the Ph SAM sixfold repeat polymer. Yet, PHC3 SAM polymerizes using just five SAMs per turn of the helical polymer rather than the typical six per turn observed for all SAM polymers reported to date. Structural analysis suggested that malleability of the PHC3 SAM would allow formation of not just the fivefold repeat structure but also possibly others. Indeed, a second PHC3 SAM polymer in a different crystal form forms a sixfold repeat polymer. These results suggest that the polymers formed by PHC3 SAM, and likely others, are dynamic. The functional consequence of the variable PHC3 SAM polymers may be to create different chromatin architectures.
Assuntos
Modelos Moleculares , Fragmentos de Peptídeos/química , Complexo Repressor Polycomb 1/química , Engenharia de Proteínas , Motivos de Aminoácidos , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Proteínas de Ligação a DNA/química , Bases de Dados de Proteínas , Proteínas de Drosophila/química , Humanos , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 1/metabolismo , Polimerização , Conformação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sequências Repetitivas de Aminoácidos , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Polyhomeotic (Ph), a member of the Polycomb Group (PcG), is a gene silencer critical for proper development. We present a previously unrecognized way of controlling Ph function through modulation of its sterile alpha motif (SAM) polymerization leading to the identification of a novel target for tuning the activities of proteins. SAM domain containing proteins have been shown to require SAM polymerization for proper function. However, the role of the Ph SAM polymer in PcG-mediated gene silencing was uncertain. Here, we first show that Ph SAM polymerization is indeed required for its gene silencing function. Interestingly, the unstructured linker sequence N-terminal to Ph SAM can shorten the length of polymers compared with when Ph SAM is individually isolated. Substituting the native linker with a random, unstructured sequence (RLink) can still limit polymerization, but not as well as the native linker. Consequently, the increased polymeric Ph RLink exhibits better gene silencing ability. In the Drosophila wing disc, Ph RLink expression suppresses growth compared with no effect for wild-type Ph, and opposite to the overgrowth phenotype observed for polymer-deficient Ph mutants. These data provide the first demonstration that the inherent activity of a protein containing a polymeric SAM can be enhanced by increasing SAM polymerization. Because the SAM linker had not been previously considered important for the function of SAM-containing proteins, our finding opens numerous opportunities to manipulate linker sequences of hundreds of polymeric SAM proteins to regulate a diverse array of intracellular functions.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila/crescimento & desenvolvimento , Drosophila/metabolismo , Nucleoproteínas/química , Nucleoproteínas/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Proteínas de Ligação a DNA/genética , Drosophila/química , Drosophila/genética , Proteínas de Drosophila/genética , Regulação da Expressão Gênica no Desenvolvimento , Inativação Gênica , Dados de Sequência Molecular , Nucleoproteínas/genética , Complexo Repressor Polycomb 1 , Polimerização , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
Polycomb-group RING finger homologs (PCGF1, PCGF2, PCGF3, PCGF4, PCGF5, and PCGF6) are critical components in the assembly of distinct Polycomb repression complex 1 (PRC1)-related complexes. Here, we identify a protein interaction domain in BCL6 corepressor, BCOR, which binds the RING finger- and WD40-associated ubiquitin-like (RAWUL) domain of PCGF1 (NSPC1) and PCGF3 but not of PCGF2 (MEL18) or PCGF4 (BMI1). Because of the selective binding, we have named this domain PCGF Ub-like fold discriminator (PUFD). The structure of BCOR PUFD bound to PCGF1 reveals that (1) PUFD binds to the same surfaces as observed for a different Polycomb group RAWUL domain and (2) the ability of PUFD to discriminate among RAWULs stems from the identity of specific residues within these interaction surfaces. These data show the molecular basis for determining the binding preference for a PCGF homolog, which ultimately helps determine the identity of the larger PRC1-like assembly.