Using Shifts in Amino Acid Frequency and Substitution Rate to Identify Latent Structural Characters in Base-Excision Repair Enzymes

Protein evolution includes the birth and death of structural motifs. For example, a zinc finger or a salt bridge may be present in some, but not all, members of a protein family. We propose that such transitions are manifest in sequence phylogenies as concerted shifts in substitution rates of amino acids that are neighbors in a representative structure. First, we identified rate shifts in a quartet from the Fpg/Nei family of base excision repair enzymes using a method developed by Xun Gu and coworkers. We found the shifts to be spatially correlated, more precisely, associated with a flexible loop involved in bacterial Fpg substrate specificity. Consistent with our result, sequences and structures provide convincing evidence that this loop plays a very different role in other family members. Second, then, we developed a method for identifying latent protein structural characters (LSC) given a set of homologous sequences based on Gu's method and proximity in a high-resolution structure. Third, we identified LSC and assigned states of LSC to clades within the Fpg/Nei family of base excision repair enzymes. We describe seven LSC; an accompanying Proteopedia page (http://proteopedia.org/wiki/index.php/Fpg_Nei_Protein_Family) describes these in greater detail and facilitates 3D viewing. The LSC we found provided a surprisingly complete picture of the interaction of the protein with the DNA capturing familiar examples, such as a Zn finger, as well as more subtle interactions. Their preponderance is consistent with an important role as phylogenetic characters. Phylogenetic inference based on LSC provided convincing evidence of independent losses of Zn fingers. Structural motifs may serve as important phylogenetic characters and modeling transitions involving structural motifs may provide a much deeper understanding of protein evolution

Genomic Ancestry, CYP2D6, CYP2C9, and CYP2C19 Among Latin Americans

We present the distribution of CYP2D6, CYP2C9, and CYP2C19 variants and predicted phenotypes in 33 native and admixed populations from Ibero-America (n > 6,000) in the context of genetic ancestry (n = 3,387). Continental ancestries are the major determinants of frequencies of the increased-activity allele CYP2C19*17 and CYP2C19 gUMs (negatively associated with Native American ancestry), decreased-activity alleles CYP2D6*41 and CYP2C9*2 (positively associated with European ancestry), and decreased-activity alleles CYP2D6*17 and CYP2D6*29 (positively associated with African ancestry). For the rare alleles, CYP2C9*2 and CYPC19*17, European admixture accounts for their presence in Native American populations, but rare alleles CYP2D6*5 (null-activity), CYP2D6-multiplication alleles (increased activity), and CYP2C9*3 (decreased-activity) were present in the pre-Columbian Americas. The study of a broad spectrum of Native American populations from different ethno-linguistic groups show how autochthonous diversity shaped the distribution of pharmaco-alleles and give insights on the prevalence of clinically relevant phenotypes associated with drugs, such as paroxetine, tamoxifen, warfarin, and clopidogrel

LSCs supply or stabilize residues that participate in enzyme-DNA interactions.

<p>Top) Amino acids side chains associated with LSC 1–6 are shown in the context of the protein backbone, DNA backbone, damaged nucleotide, opposite nucleotide, and Zn ion <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025246#pone.0025246-Fromme1" target="_blank">[67]</a>. The green residues in both the structure (top) and the diagram (bottom) correspond to first-shell amino acids conserved in the entire family: R264 (contained in LSC6), N174 (stabilized by LSC1), and K60 (stabilized by LSC3/LSC2) stabilize the phosphate of the damaged base, and P2, E3 and are part of the catalytic residues <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025246#pone.0025246-Gilboa1" target="_blank">[97]</a>. The helix containing P2 and E3 may be stabilized by LSC2 as well. The enzyme everts the damage, and an intercalation loop (LSC4) fills the void and makes contact with the opposite base <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025246#pone.0025246-Coste1" target="_blank">[68]</a>. The damage itself in BaFpg1 is recognized by a recognition complex <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025246#pone.0025246-Fromme1" target="_blank">[67]</a>. Other important residues not included here include H74 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025246#pone.0025246-Gilboa1" target="_blank">[97]</a> and E6 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025246#pone.0025246-Kropachev1" target="_blank">[48]</a>. A DNA binding residue not discussed in the literature corresponds to Tyr242 (part of LSC5).</p

Rate variation does not differ dramatically between replicate Proteobacterium, Actinomycete, or Eukaryote organism tree topologies.

<p>Each column corresponds to one of the three organismal phylogenies. Each entry in a column (paired blue and green bars) represents an instance of the organismal phylogeny in the Fpg/Nei family protein phylogeny. The blue bars correspond to the number of substitutions from the last common ancestor (LCA) of each replicate tree to the present while the green bars correspond to the number of substitutions from the LCA of the phylogeny of replicate trees to the LCA of the each replicate tree.</p

Multiple States of an LSC: Two solutions to the same problem.

<p>An LSC can have multiple states. A) State of LSC1 in the B. stearothermophilus MutM structure <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025246#pone.0025246-Fromme1" target="_blank">[67]</a>. N174 (in pink), part of the helix-two-turn-helix (H2TH) motif along with two other amino acids (including the key amino acid R264, in blue) functions in the orientation and kinking of the DNA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025246#pone.0025246-Zharkov2" target="_blank">[70]</a>. K160 (blue) helps keep the proper arrangement between the zinc finger and the H2TH <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025246#pone.0025246-Sugahara1" target="_blank">[69]</a>. B) Sequence logos for the each of the nine LSC1 amino acids in each of the three clades as well as MvNei1. Column headings indicate the aligned position in both the B. stearothermophilus MutM and E. coli Nei sequences. The sequence logos associated with 1R2Y K160 suggest that in three of the nine clades (BaFpg1, BaFpg2 and PFNei) the arrangement between the zinc finger and the H2TH is stabilized by a lysine in the same manner as in the B. stearothermophilus MutM protein. C) State of LSC1 in the E. coli Nei structure (62, PDB 1K3W). R171 hydrogen bonds to the other beta-sheet of the zinc-finger, presumably playing a role analogous to 1R2Y K160, which originates on a different helix. The sequence logos associated with R171 suggests that in six subfamilies (AcNei1 and AcNei2, PrNei and all vertebrate subfamilies), the arrangement between the zinc finger and the H2TH is maintained by an arginine or lysine in the same manner as in the E. coli Nei protein. For the subfamilies of BaFpg1 and PrNei, sites 160 and 266 are a type I, 174 and 264 are a type 0, and the rest are type II.</p

Coefficient of Type I (above diagonal) and Type II (below diagonal) functional divergence for Fpg/Nei clades.

<p>Coefficient of Type I (above diagonal) and Type II (below diagonal) functional divergence for Fpg/Nei clades.</p

Substitution rates of individual aligned amino acid positions can differ between clades of orthologs.

<p>Substitution rates of individual aligned amino acid positions can differ between clades of orthologs from actinomycetes (left, Pearson correlation 0.47) or eukaryotes (right, 0.19). Each axis reflects amino acid variation rate in one of the replicate organism trees described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025246#pone-0025246-g004" target="_blank">Figure 4</a>. Each point is an aligned amino acid sequence position. Sites that have experienced a rate-shift (Type I) are green while those that exhibit an amino acid frequency-shift (Type II) are red.</p