Diversification of DNA-Binding Specificity by Permissive and Specificity-Switching Mutations in the ParB/Noc Protein Family

Abstract

Summary Specific interactions between proteins and DNA are essential to many biological processes. Yet, it remains unclear how the diversification in DNA-binding specificity was brought about, and the mutational paths that led to changes in specificity are unknown. Using a pair of evolutionarily related DNA-binding proteins, each with a different DNA preference (ParB [Partitioning Protein B] and Noc [Nucleoid Occlusion Factor], which both play roles in bacterial chromosome maintenance), we show that specificity is encoded by a set of four residues at the protein-DNA interface. Combining X-ray crystallography and deep mutational scanning of the interface, we suggest that permissive mutations must be introduced before specificity-switching mutations to reprogram specificity and that mutational paths to new specificity do not necessarily involve dual-specificity intermediates. Overall, our results provide insight into the possible evolutionary history of ParB and Noc and, in a broader context, might be useful for understanding the evolution of other classes of DNA-binding proteins.

The CTPase activity of ParB acts as a timing mechanism to control the dynamics and function of prokaryotic DNA partition complexes

“…Next, we reinvestigated the structural consequences of parS binding ( Figure 2C ). Consistent with our previous results (Osorio-Valeriano et al, 2019), we detected a strong protection of the DNA-binding domain, especially in regions previously shown to interact directly with the associated DNA molecule (Chen et al, 2015; Jalal et al, 2020b; Sanchez et al, 2013). In addition, we also observed the deprotection of regions in the NB domain, including helices α3 and α4 and sheets β1-β4.…”

“…This result may again indicate the release of helix α4 from the DNA-binding domain, triggered by parS -induced conformational changes in helices α5 and α6. Notably, in crystal structures of ParB· parS complexes (Chen et al, 2015; Jalal et al, 2020b), helices α4-α6 still adopt an arrangement similar to that in the apo state ( Figure S3C ). We hypothesize that parS binding weakens the interactions in this region but does not fully abolish them.…”

CTP regulates membrane-binding activity of the nucleoid occlusion protein Noc

“…Finally, evolution is replete with examples of functional domains being adapted to diversify functions of proteins. Gene encoding Noc resulted from gene duplication and neo-functionalization from parB [15][16][17] , our work furthers the understanding of how a CTP switch has been adapted to a new function, and hence might have implications in understanding biological innovations by evolution.…”

“…The dashed line demarcates the NTD from the DNA-binding domain (DBD). Helix α8 at the DBD is the recognition helix that contributes to the specific recognition of the NBS site 17,24 . (B) The membrane-targeting amphipathic helix α1.…”

“…In Firmicutes, the nucleoid occlusion protein Noc is a paralog of ParB [15][16][17] ; however, the role of Noc is different from that of a canonical ParB [18][19][20] . Noc helps to direct the assembly of the cell division machinery towards the middle of a dividing cell where the concentration of chromosomal DNA (the nucleoid) is the least, thus ensuring a binary cell division 18,[21][22][23] .…”