The orderly deposition of histones onto DNA is mediated by conserved assembly complexes, including chromatin assembly factor-1 (CAF-1) and the Hir proteins . CAF-1 and the Hir proteins operate in distinct but functionally overlapping histone deposition pathways in vivo . The Hir proteins and CAF-1 share a common partner, the highly conserved histone H3/H4 binding protein Asf1, which binds the middle subunit of CAF-1 as well as to Hir proteins . Asf1 binds to newly synthesized histones H3/H4 , and this complex stimulates histone deposition by CAF-1 . In yeast, Asf1 is required for the contribution of the Hir proteins to gene silencing . Here, we demonstrate that Hir1, Hir2, Hir3, and Hpc2 comprise the HIR complex, which copurifies with the histone deposition protein Asf1. Together, the HIR complex and Asf1 deposit histones onto DNA in a replication-independent manner. Histone deposition by the HIR complex and Asf1 is impaired by a mutation in Asf1 that inhibits HIR binding. These data indicate that the HIR complex and Asf1 proteins function together as a conserved eukaryotic pathway for histone replacement throughout the cell cycle.
The histone chaperone Rtt106 binds histone H3 acetylated at lysine 56 (H3K56ac) and facilitates nucleosome assembly during several molecular processes. Both the structural basis of this modificationspecific recognition and how this recognition informs Rtt106 function are presently unclear. Guided by our crystal structure of Rtt106, we identified two regions on its double-pleckstrin homology domain architecture that mediated histone binding. When histone binding was compromised, Rtt106 localized properly to chromatin but failed to deliver H3K56ac, leading to replication and silencing defects. By mutating analogous regions in the structurally homologous chromatin-reorganizer Pob3, we revealed a conserved histone-binding function for a basic patch found on both proteins. In contrast, a loop connecting two β-strands was required for histone binding by Rtt106 but was dispensable for Pob3 function. Unlike Rtt106, Pob3 histone binding was modification-independent, implicating the loop of Rtt106 in H3K56ac-specific recognition in vivo. Our studies described the structural origins of Rtt106 function, identified a conserved histone-binding surface, and defined a critical role for Rtt106:H3K56ac-binding specificity in silencing and replication-coupled nucleosome turnover.histone acetylation | yFACT | CAF-1 | Sir | Saccharomyces cerevisiae
Summaryβ-lactamase/β-lactamase Inhibitor Protein (BLIP) complexes are emerging as a well characterized experimental model system for studying protein-protein interactions. β-lactamases are enzymes that catalyze the hydrolysis of β-lactam antibiotics. BLIP is a 165 amino acid protein that inhibits several class A β-lactamases with a wide range of affinities: pM affinity for K1; nM affinity for TEM-1, SME-1, and BlaI but only µM affinity for SHV-1 β-lactamase. The large differences in affinity coupled with the availability of extensive mutagenesis data and high resolution crystal structures for the TEM-1/BLIP and SHV-1/BLIP complexes make them attractive systems for the further development of protein engineering and computational design methodologies. We used EGAD, a physics-based computational design program, to redesign BLIP with the goal of increasing affinity for SHV-1. The resulting designed sequences are highly similar to wildtype, with the exception of BLIP residues surrounding β-lactamase position 104. Interestingly, this residue is a known specificity determinant between TEM-1 and SHV-1. Characterization of several of the designs and point mutants revealed that in all cases, the mutations stabilize the interface by 10 to 1000 fold relative to wildtype BLIP. The calculated changes in binding affinity for the mutants were within a mean absolute error of 0.87 kcal/mol from the experimental values, and comparison of calculated and experimental values for a set of 30 SHV-1/BLIP complexes yielded a correlation coefficient of 0.77. Although binding specificity for SHV-1 versus TEM-1 was not explicitly considered in the design process, two of the BLIP variants exhibit a small specificity switch. Structures of the two highest affinity complexes, SHV-1/BLIP (E73M) and SHV-1/BLIP (E73M, S130K, S146M), are presented at 1.7 Å resolution. While the predicted structures have much in common with the experimentally determined structures, they do not coincide perfectly; in particular a salt bridge between SHV-1 D104 and BLIP K74 is observed in
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