Engineering mono- and multi-valent inhibitors on a modular scaffold.

Here we exploit the simple, ultra-stable, modular architecture of consensus-designed tetratricopeptide repeat proteins (CTPRs) to create a platform capable of displaying both single as well as multiple functions and with diverse programmable geometrical arrangements by grafting non-helical short linear binding motifs (SLiMs) onto the loops between adjacent repeats. As proof of concept, we built synthetic CTPRs to bind and inhibit the human tankyrase proteins (hTNKS), which play a key role in Wnt signaling and are upregulated in cancer. A series of mono-valent and multi-valent hTNKS binders was assembled. To fully exploit the modular scaffold and to further diversify the multi-valent geometry, we engineered the binding modules with two different formats, one monomeric and the other trimeric. We show that the designed proteins are stable, correctly folded and capable of binding to and inhibiting the cellular activity of hTNKS leading to downregulation of the Wnt pathway. Multivalency in both the CTPR protein arrays and the hTNKS target results in the formation of large macromolecular assemblies, which can be visualized both in vitro and in the cell. When delivered into the cell by nanoparticle encapsulation, the multivalent CTPR proteins displayed exceptional activity. They are able to inhibit Wnt signaling where small molecule inhibitors have failed to date. Our results point to the tremendous potential of the CTPR platform to exploit a range of SLiMs and assemble synthetic binding molecules with built-in multivalent capabilities and precise, pre-programmed geometries.

Histone H2A-H2B binding by Pol α in the eukaryotic replisome contributes to the maintenance of repressive chromatin.

The eukaryotic replisome disassembles parental chromatin at DNA replication forks, but then plays a poorly understood role in the re-deposition of the displaced histone complexes onto nascent DNA. Here, we show that yeast DNA polymerase α contains a histone-binding motif that is conserved in human Pol α and is specific for histones H2A and H2B. Mutation of this motif in budding yeast cells does not affect DNA synthesis, but instead abrogates gene silencing at telomeres and mating-type loci. Similar phenotypes are produced not only by mutations that displace Pol α from the replisome, but also by mutation of the previously identified histone-binding motif in the CMG helicase subunit Mcm2, the human orthologue of which was shown to bind to histones H3 and H4. We show that chromatin-derived histone complexes can be bound simultaneously by Mcm2, Pol α and the histone chaperone FACT that is also a replisome component. These findings indicate that replisome assembly unites multiple histone-binding activities, which jointly process parental histones to help preserve silent chromatin during the process of chromosome duplication.

The basic keratin 10-binding domain of the virulence-associated pneumococcal serine-rich protein PsrP adopts a novel MSCRAMM fold.

Streptococcus pneumoniae is a major human pathogen, and a leading cause of disease and death worldwide. Pneumococcal invasive disease is triggered by initial asymptomatic colonization of the human upper respiratory tract. The pneumococcal serine-rich repeat protein (PsrP) is a lung-specific virulence factor whose functional binding region (BR) binds to keratin-10 (KRT10) and promotes pneumococcal biofilm formation through self-oligomerization. We present the crystal structure of the KRT10-binding domain of PsrP (BR187-385) determined to 2.0 Å resolution. BR187-385 adopts a novel variant of the DEv-IgG fold, typical for microbial surface components recognizing adhesive matrix molecules adhesins, despite very low sequence identity. An extended ß-sheet on one side of the compressed, two-sided barrel presents a basic groove that possibly binds to the acidic helical rod domain of KRT10. Our study also demonstrates the importance of the other side of the barrel, formed by extensive well-ordered loops and stabilized by short ß-strands, for interaction with KRT10.