Cyp33 binds AU-rich RNA motifs via an extended interface that competitively disrupts the gene repressive Cyp33-MLL1 interaction in vitro.

Cyp33 is an essential human cyclophilin prolyl isomerase that plays myriad roles in splicing and chromatin remodeling. In addition to a canonical cyclophilin (Cyp) domain, Cyp33 contains an RNA-recognition motif (RRM) domain, and RNA-binding triggers proline isomerase activity. One prominent role for Cyp33 is through a direct interaction with the mixed lineage leukemia protein 1 (MLL1, also known as KMT2A) complex, which is a histone methyltransferase that serves as a global regulator of human transcription. MLL activity is regulated by Cyp33, which isomerizes a key proline in the linker between the PHD3 and Bromo domains of MLL1, acting as a switch between gene activation and repression. The direct interaction between MLL1 and Cyp33 is critical, as deletion of the MLL1-PHD3 domain responsible for this interaction results in oncogenesis. The Cyp33 RRM is central to these activities, as it binds both the PHD3 domain and RNA. To better understand how RNA binding drives the action of Cyp33, we performed RNA-SELEX against full-length Cyp33 accompanied by deep sequencing. We have identified an enriched Cyp33 binding motif (AAUAAUAA) broadly represented in the cellular RNA pool as well as tightly binding RNA aptamers with affinities comparable and competitive with the Cyp33 MLL1-PHD3 interaction. RNA binding extends beyond the canonical RRM domain, but not to the Cyp domain, suggesting an indirect mechanism of interaction. NMR chemical shift mapping confirms an overlapping, but not identical, interface on Cyp33 for RNA and PHD3 binding. This finding suggests RNA can disrupt the gene repressive Cyp33-MLL1 complex providing another layer of regulation for chromatin remodeling by MLL1.

Discrimination against RNA Backbones by a ssDNA Binding Protein.

Pot1 is the shelterin component responsible for the protection of the single-stranded DNA (ssDNA) overhang at telomeres in nearly all eukaryotic organisms. The C-terminal domain of the DNA-binding domain, Pot1pC, exhibits non-specific ssDNA recognition, achieved through thermodynamically equivalent alternative binding conformations. Given this flexibility, it is unclear how specificity for ssDNA over RNA, an activity required for biological function, is achieved. Examination of the ribose-position specificity of Pot1pC shows that ssDNA specificity is additive but not uniformly distributed across the ligand. High-resolution structures of several Pot1pC complexes with RNA-DNA chimeric ligands reveal Pot1pC discriminates against RNA by utilizing non-compensatory binding modes that feature significant rearrangement of the binding interface. These alternative conformations, accessed through both ligand and protein flexibility, recover much, but not all, of the binding energy, leading to the observed reduction in affinities. These findings suggest that intermolecular interfaces are remarkably sophisticated in their tuning of specificity toward flexible ligands.

Tying up the Ends: Plasticity in the Recognition of Single-Stranded DNA at Telomeres.

Telomeres terminate nearly exclusively in single-stranded DNA (ssDNA) overhangs comprised of the G-rich 3' end. This overhang varies widely in length from species to species, ranging from just a few bases to several hundred nucleotides. These overhangs are not merely a remnant of DNA replication but rather are the result of complex further processing. Proper management of the telomeric overhang is required both to deter the action of the DNA damage machinery and to present the ends properly to the replicative enzyme telomerase. This Current Topic addresses the biochemical and structural features used by the proteins that manage these variable telomeric overhangs. The Pot1 protein tightly binds the single-stranded overhang, preventing DNA damage sensors from binding. Pot1 also orchestrates the access of telomerase to that same substrate. The remarkable plasticity of the binding interface exhibited by the Schizosaccharomyces pombe Pot1 provides mechanistic insight into how these roles may be accomplished, and disease-associated mutations clustered around the DNA-binding interface in the hPOT1 highlight the importance of this function. The budding yeast Cdc13-Stn1-Ten1, a telomeric RPA complex closely associated with telomere function, also interacts with ssDNA in a fashion that allows degenerate sequences to be recognized. A related human complex composed of hCTC1, hSTN1, and hTEN1 has recently emerged with links to both telomere maintenance and general DNA replication and also exhibits mutations associated with telomere pathologies. Overall, these sequence-specific ssDNA binders exhibit a range of recognition properties that allow them to perform their unique biological functions.

Less is more: structures of difficult targets with minimal constraints.

By merging recent experimental and computational methodology advances, resolution-adapted structural recombination Rosetta has emerged as a powerful strategy for solving the structure of traditionally challenging targets. In this issue of Structure, Sgourakis and colleagues solve the structure of one such target, the immunoevasin protein m04, using this approach.