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SP_0782 from Streptococcus pneumoniae is a dimeric PC4-like protein binding single-stranded DNA (ssDNA), and is potentially involved in maintenance of genome stability and natural transformation. SP_0782 binds with different lengths of ssDNA in various patterns through accommodating nucleotides differently in its two DNA-binding regions (DBRs). Here, we report the characterization of a novel site, leucine 20 (L20), which is not located in the DBRs but impairs the DNA binding when mutated to alanine (L20A). The L20A mutation markedly reduced the DNA-binding affinity of SP_0782 for ssDNA dT19G1, and affected the formation of high-order SP_0782:dT19G1 complexes. The side chain of L20 shows interactions with several residues at the backside of the DBRs in apo SP_0782 structure, and the L20A mutation led to a change of circular dichroism (CD) spectrum and broad chemical shift perturbations (CSPs) in NMR spectrum compared with the wild type. The most affected residues in NMR spectrum included F39 and R49 located in DBR2, as well as K60 in DBR1, which was suggested to be important for cooperative binding of ssDNA by the two subunits in SP_0782 dimer. Thus, the L20A mutation caused a local conformational change of SP_0782, which exerted an indirect effect on the DNA-binding interface and therefore impaired the affinity for ssDNA dT19G1. Interestingly, this L20 site is conserved in bacterial but not eukaryotic PC4-like proteins, suggesting an evolutionary divergence. This study provides an insight into the structure-function relationship of SP_0782, and an amino-acid site probably targeted for inhibiting bacteria selectively. • L20A mutation markedly impairs the binding affinity of SP_0782 for dT19G1. • L20A mutation changes the local conformation of SP_0782. • L20A mutation exerts an indirect effect on the DNA-binding interface of SP_0782. • The L20 site is conserved in bacterial but not eukaryotic PC4-like proteins. [ABSTRACT FROM AUTHOR]

Abstract Human protein TGIF1 is an essential regulator of cell fate with broad roles in different tissues, and has been implicated in holoprosencephaly (HPE) and many cancers. The function of TGIF1 in transcriptional regulation depends on its three-amino acid loop extension (TALE) type of homeodomain (HD). Two missense mutations that led to P192A and R219C substitutions in TGIF1-HD were previously found in HPE patients and suggested to be the causes for these cases. However, how these mutations affected TGIF1 function has not been investigated from a structural view. Here, we investigated the roles of P192 and R219 in TGIF1-HD structure packing through determining the NMR structure of TGIF1-HD. Surprisingly, P192 and R219 were found to play roles in packing α1 and α2 to α3 together with A190 and F215 through side-chain interactions. Circular dichroism (CD) showed that P192A and R219C mutants displayed structural change and less folding compared with wild-type TGIF1-HD, and 1H-15N HSQC spectrum of P192A mutant exhibited chemical shift perturbations in all three helices of TGIF1-HD. Thus, it is suggested that P192A and R219C mutations led to structure disturbances of TGIF1-HD, which subsequently reduced the DNA-binding affinity of TGIF1-HD by 23-fold and 10-fold respectively, as revealed by the isothermal titration calorimetry (ITC) experiments. Our study provides structural insights of the probable pathogenesis mechanism of two TGIF1-related HPE cases, and evidences for the roles of P192 and R219 in HD folding. Highlights • HPE-related mutations P129A and R219C impair DNA affinity of TGIF1 homeodomain. • P192 and R219 play roles in packing α1 and α2 to α3 in TGIF1 homeodomain. • P129A and R219C lead to structural change and less folding of TGIF1 homeodomain. • P192A results in chemical shift perturbations in all three helices of TGIF1 homeodomain. [ABSTRACT FROM AUTHOR]