Abstract
BackgroundTotal sequence decomposition, using the web-based MASIA tool, identifies areas of conservation in aligned protein sequences. By structurally annotating these motifs, the sequence can be parsed into individual building blocks, molecular legos ("molegos"), that can eventually be related to function. Here, the approach is applied to the apurinic/apyrimidinic endonuclease (APE) DNA repair proteins, essential enzymes that have been highly conserved throughout evolution. The APEs, DNase-1 and inositol 5'-polyphosphate phosphatases (IPP) form a superfamily that catalyze metal ion based phosphorolysis, but recognize different substrates.ResultsMASIA decomposition of APE yielded 12 sequence motifs, 10 of which are also structurally conserved within the family and are designated as molegos. The 12 motifs include all the residues known to be essential for DNA cleavage by APE. Five of these molegos are sequentially and structurally conserved in DNase-1 and the IPP family. Correcting the sequence alignment to match the residues at the ends of two of the molegos that are absolutely conserved in each of the three families greatly improved the local structural alignment of APEs, DNase-1 and synaptojanin. Comparing substrate/product binding of molegos common to DNase-1 showed that those distinctive for APEs are not directly involved in cleavage, but establish protein-DNA interactions 3' to the abasic site. These additional bonds enhance both specific binding to damaged DNA and the processivity of APE1.ConclusionA modular approach can improve structurally predictive alignments of homologous proteins with low sequence identity and reveal residues peripheral to the traditional "active site" that control the specificity of enzymatic activity.
Highlights
Total sequence decomposition, using the web-based MASIA tool, identifies areas of conservation in aligned protein sequences
Our analysis identified 5 molegos that are common to the DNase 1, inositol 5'-polyphosphate phosphatases (IPP) and apyrimidinic endonuclease (APE) families, which roughly correspond to areas of sequence similarity identified previously [31,32]
The motifs identified in the APE family could be structurally annotated using crystal data to derive molegos, words in the protein sequence that correlate with structural elements
Summary
Total sequence decomposition, using the web-based MASIA tool, identifies areas of conservation in aligned protein sequences. By structurally annotating these motifs, the sequence can be parsed into individual building blocks, molecular legos ("molegos"), that can eventually be related to function. The approach is applied to the apurinic/apyrimidinic endonuclease (APE) DNA repair proteins, essential enzymes that have been highly conserved throughout evolution. While many endonucleases share a common active site that is highly conserved across many subfamilies, identifying residues that control substrate specificity re-. We have chosen to illustrate this approach using the multifunctional family of DNA repair proteins, the apurinic/apyrimidinic endonucleases (APEs), which have a clearly defined bacterial ancestor, E. coli exonuclease III (ExoIII), and are distantly related to several enzymes with varying substrate specificity
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