HDAC6 and Ubp-M BUZ Domains Recognize Specific C-Terminal Sequences of Proteins

摘要

The BUZ/Znf-UBP domain is a proteinmodule found in the cytoplasmic deacetylaseHDAC6, E3nubiquitin ligase BRAP2/IMP, and a subfamily of ubiquitin-specific proteases. Although several BUZndomains have been shown to bind ubiquitin with high affinity by recognizing its C-terminal sequencen(RLRGG-COOH), it is currently unknown whether the interaction is sequence-specific or whether the BUZndomains are capable of binding to proteins other than ubiquitin. In this work, the BUZ domains of HDAC6nand Ubp-M were subjected to screening against a one-bead-one-compound (OBOC) peptide library thatnexhibited randompeptide sequences with free C-termini. Sequence analysis of the selected binding peptides asnwell as alanine scanning studies revealed that the BUZ domains require a C-terminal Gly-Gly motif fornbinding. At the more N-terminal positions, the two BUZ domains have distinct sequence specificities,nallowing them to bind to different peptides and/or proteins. A database search of the human proteome on thenbasis of the BUZ domain specificities identified 11 and 24 potential partner proteins for Ubp-Mand HDAC6nBUZ domains, respectively. Peptides corresponding to the C-terminal sequences of four of the predictednbinding partners (FBXO11, histone H4, PTOV1, and FAT10) were synthesized and tested for binding to thenBUZ domains by fluorescence polarization. All four peptides bound to the HDAC6 BUZ domain with lownmicromolar KD values and less tightly to the Ubp-MBUZ domain. Finally, in vitro pull-down assays showednthat the Ubp-M BUZ domain was capable of binding to the histone H3-histone H4 tetramer proteinncomplex. Our results suggest that BUZ domains are sequence-specific protein-binding modules, with eachnBUZ domain potentially binding to a different subset of proteins.