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Poly(C) Binding Protein Binding Affinity and Specificity for C-rich Oligonucleotides
thesis
posted on 2017-03-16, 01:20 authored by Christopher SzetoPoly(C) Binding
Proteins (PCBPs) are RNA-binding proteins that interact with pre-mRNAs and
mRNAs for the purpose of regulating post-transcriptional events. PCBPs have
high affinity and specificity for poly-cytosine in single stranded
deoxyribonucleic acid (ssDNA) and single stranded ribonucleic acid (ssRNA).
This allows PCBPs to interact with C-rich RNA sequences to achieve functions
such as pre-mRNA splicing, mRNA stability and mRNA translational activation and
silencing. Additionally, PCBPs have also been revealed to be hijacked from host
cell function to aid in viral protein translation and the replication of
positive-strand picornaviruses such as the poliovirus.
PCBPs consist of three hnRNP K (KH) domains that work in tandem to recognise and bind to C-rich RNA structures. The way in which full length PCBPs engage RNA structures would enable the understanding of protein-oligonucleotide complexes formed during processes of post-transcriptional regulation. However, structures of multi-KH domain PCBPs in complex with oligonucleotide have never been solved. Instead, single PCBP KH1 domains have been solved in complex with oligonucleotide. Three solved structures revealed that the KH domain consists of a hydrophobic core with a number of charged amino acids at the site of the nucleotide binding groove that can accommodate up to 4 nucleotide bases. Each of these structures shows that KH1 recognises a different tetra-nucleotide motif (ACCC, CCCT and CCCC). These structures are unable to explain the molecular basis for nucleotide recognition at the 1st and 4th positions of the tetra-nucleotide motif.
Binding studies using Surface Plasmon Resonance and Fluorescence Anisotropy (FA) were used to show that KH1 bound to the CCCC motif with the highest affinity; and was 2-fold higher than the CCCT motif and 4-fold higher than ACCC. This suggested that preferential binding for cytosine exists at the 1st and/or 4th positions. Using Molecular Dynamics (MD), each of the KH1-oligonucleotide complexes were modelled in silico to predict the interactions that might contribute to binding affinity and specificity for cytosine. My in silico investigations predicted 4 amino acids: D82, R57, R40 and E51 (at the 1st, 2nd, 3rd and 4th positions respectively). This was followed up experimentally by mutating each of these residues to alanine using site-directed mutagenesis. Their binding affinities were measured using FA and results revealed that D82 does not contribute to binding affinity or specificity for cytosine at the 1st position. However, the residues R57, R40 and E51 are responsible for KH1’s recognition of cytosine triplets at the 2nd, 3rd and 4th nucleotide binding positions.
Although KH1 interactions that underlie affinity and specificity for C-rich DNA were established, the ways in which full length PCBPs bind to target RNAs were still unclear. Using binding and structural studies, it was discovered that the KH2 domain, by itself, possesses extremely weak binding affinity to C-rich DNA; however, when together in a construct with KH1, it is able to support the KH1 domain in binding C-rich DNA in a synergistic manner. These results suggest that an ordered arrangement of KH domains may exist. Crystals of KH1-KH2 proteins were successfully prepared; however, did not diffract at high enough resolution for structural determination. Together, these studies provide a framework for further structural studies to uncover the way full length PCBPs bind to biologically relevant RNA targets.
PCBPs consist of three hnRNP K (KH) domains that work in tandem to recognise and bind to C-rich RNA structures. The way in which full length PCBPs engage RNA structures would enable the understanding of protein-oligonucleotide complexes formed during processes of post-transcriptional regulation. However, structures of multi-KH domain PCBPs in complex with oligonucleotide have never been solved. Instead, single PCBP KH1 domains have been solved in complex with oligonucleotide. Three solved structures revealed that the KH domain consists of a hydrophobic core with a number of charged amino acids at the site of the nucleotide binding groove that can accommodate up to 4 nucleotide bases. Each of these structures shows that KH1 recognises a different tetra-nucleotide motif (ACCC, CCCT and CCCC). These structures are unable to explain the molecular basis for nucleotide recognition at the 1st and 4th positions of the tetra-nucleotide motif.
Binding studies using Surface Plasmon Resonance and Fluorescence Anisotropy (FA) were used to show that KH1 bound to the CCCC motif with the highest affinity; and was 2-fold higher than the CCCT motif and 4-fold higher than ACCC. This suggested that preferential binding for cytosine exists at the 1st and/or 4th positions. Using Molecular Dynamics (MD), each of the KH1-oligonucleotide complexes were modelled in silico to predict the interactions that might contribute to binding affinity and specificity for cytosine. My in silico investigations predicted 4 amino acids: D82, R57, R40 and E51 (at the 1st, 2nd, 3rd and 4th positions respectively). This was followed up experimentally by mutating each of these residues to alanine using site-directed mutagenesis. Their binding affinities were measured using FA and results revealed that D82 does not contribute to binding affinity or specificity for cytosine at the 1st position. However, the residues R57, R40 and E51 are responsible for KH1’s recognition of cytosine triplets at the 2nd, 3rd and 4th nucleotide binding positions.
Although KH1 interactions that underlie affinity and specificity for C-rich DNA were established, the ways in which full length PCBPs bind to target RNAs were still unclear. Using binding and structural studies, it was discovered that the KH2 domain, by itself, possesses extremely weak binding affinity to C-rich DNA; however, when together in a construct with KH1, it is able to support the KH1 domain in binding C-rich DNA in a synergistic manner. These results suggest that an ordered arrangement of KH domains may exist. Crystals of KH1-KH2 proteins were successfully prepared; however, did not diffract at high enough resolution for structural determination. Together, these studies provide a framework for further structural studies to uncover the way full length PCBPs bind to biologically relevant RNA targets.
