More Like this

1. Alignment-Integrated Reconstruction of Ancestral Sequences Improves Accuracy

   https://doi.org/10.1093/gbe/evaa164  

   Aadland, Kelsey ; Kolaczkowski, Bryan ( September 2020 , Genome Biology and Evolution)

   dos Reis, Mario (Ed.)

   Abstract Ancestral sequence reconstruction (ASR) uses an alignment of extant protein sequences, a phylogeny describing the history of the protein family and a model of the molecular-evolutionary process to infer the sequences of ancient proteins, allowing researchers to directly investigate the impact of sequence evolution on protein structure and function. Like all statistical inferences, ASR can be sensitive to violations of its underlying assumptions. Previous studies have shown that, whereas phylogenetic uncertainty has only a very weak impact on ASR accuracy, uncertainty in the protein sequence alignment can more strongly affect inferred ancestral sequences. Here, we show that errors in sequence alignment can produce errors in ASR across a range of realistic and simplified evolutionary scenarios. Importantly, sequence reconstruction errors can lead to errors in estimates of structural and functional properties of ancestral proteins, potentially undermining the reliability of analyses relying on ASR. We introduce an alignment-integrated ASR approach that combines information from many different sequence alignments. We show that integrating alignment uncertainty improves ASR accuracy and the accuracy of downstream structural and functional inferences, often performing as well as highly accurate structure-guided alignment. Given the growing evidence that sequence alignment errors can impact the reliability of ASR studies, we recommend that future studies incorporate approaches to mitigate the impact of alignment uncertainty. Probabilistic modeling of insertion and deletion events has the potential to radically improve ASR accuracy when the model reflects the true underlying evolutionary history, but further studies are required to thoroughly evaluate the reliability of these approaches under realistic conditions. 

   more » « less
2. Topiary: Pruning the manual labor from ancestral sequence reconstruction

   https://doi.org/10.1002/pro.4551  

   Orlandi, Kona N. ; Phillips, Sophia R. ; Sailer, Zachary R. ; Harman, Joseph L. ; Harms, Michael J. ( January 2023 , Protein Science)

   Ancestral sequence reconstruction (ASR) is a powerful tool to study the evolution of proteins and thus gain deep insight into the relationships among protein sequence, structure, and function. A major barrier to its broad use is the complexity of the task: it requires multiple software packages, complex file manipulations, and expert phylogenetic knowledge. Here we introducetopiary, a software pipeline that aims to overcome this barrier. To use topiary, users prepare a spreadsheet with a handful of sequences. Topiary then: (1) Infers the taxonomic scope for the ASR study and finds relevant sequences by BLAST; (2) Does taxonomically informed sequence quality control and redundancy reduction; (3) Constructs a multiple sequence alignment; (4) Generates a maximum‐likelihood gene tree; (5) Reconciles the gene tree to the species tree; (6) Reconstructs ancestral amino acid sequences; and (7) Determines branch supports. The pipeline returns annotated evolutionary trees, spreadsheets with sequences, and graphical summaries of ancestor quality. This is achieved by integrating modern phylogenetics software (Muscle5, RAxML‐NG, GeneRax, and PastML) with online databases (NCBI and the Open Tree of Life). In this paper, we introduce non‐expert readers to the steps required for ASR, describe the specific design choices made intopiary, provide a detailed protocol for users, and then validate the pipeline using datasets from a broad collection of protein families. Topiary is freely available for download:https://github.com/harmslab/topiary.

    

   more » « less
3. Hinge-shift mechanism as a protein design principle for the evolution of β-lactamases from substrate promiscuity to specificity

   https://doi.org/10.1038/s41467-021-22089-0  

   Modi, Tushar ; Risso, Valeria A. ; Martinez-Rodriguez, Sergio ; Gavira, Jose A. ; Mebrat, Mubark D. ; Van Horn, Wade D. ; Sanchez-Ruiz, Jose M. ; Banu Ozkan, S. ( March 2021 , Nature Communications)

   TEM-1 β-lactamase degrades β-lactam antibiotics with a strong preference for penicillins. Sequence reconstruction studies indicate that it evolved from ancestral enzymes that degraded a variety of β-lactam antibiotics with moderate efficiency. This generalist to specialist conversion involved more than 100 mutational changes, but conserved fold and catalytic residues, suggesting a role for dynamics in enzyme evolution. Here, we develop a conformational dynamics computational approach to rationally mold a protein flexibility profile on the basis of a hinge-shift mechanism. By deliberately weighting and altering the conformational dynamics of a putative Precambrian β-lactamase, we engineer enzyme specificity that mimics the modern TEM-1 β-lactamase with only 21 amino acid replacements. Our conformational dynamics design thus re-enacts the evolutionary process and provides a rational allosteric approach for manipulating function while conserving the enzyme active site.

    

   more » « less
4. Conserved residues are critical for Haloferax volcanii archaeosortase catalytic activity: Implications for convergent evolution of the catalytic mechanisms of non‐homologous sortases from archaea and bacteria

   https://doi.org/10.1111/mmi.13935  

   Abdul Halim, Mohd Farid ; Rodriguez, Ronald ; Stoltzfus, Jonathan D. ; Duggin, Iain G. ; Pohlschroder, Mechthild ( March 2018 , Molecular Microbiology)

   Proper protein anchoring is key to the biogenesis of prokaryotic cell surfaces, dynamic, resilient structures that play crucial roles in various cell processes. A novel surface protein anchoring mechanism inHaloferax volcaniidepends upon the peptidase archaeosortase A (ArtA) processing C‐termini of substrates containing C‐terminal tripartite structures and anchoring mature substrates to the cell membrane via intercalation of lipid‐modified C‐terminal amino acid residues. While this membrane protein lacks clear homology to soluble sortase transpeptidases of Gram‐positive bacteria, which also process C‐termini of substrates whose C‐terminal tripartite structures resemble those of ArtA substrates, archaeosortases do contain conserved cysteine, arginine and arginine/histidine/asparagine residues, reminiscent of His‐Cys‐Arg residues of sortase catalytic sites. The study presented here shows that ArtA^WT‐GFP expressedin transcomplements ΔartAgrowth and motility phenotypes, while alanine substitution mutants, Cys¹⁷³(C173A), Arg²¹⁴(R214A) or Arg²⁵³(R253A), and the serine substitution mutant for Cys¹⁷³(C173S), fail to complement these phenotypes. Consistent with sortase active site replacement mutants, ArtA^C173A‐GFP, ArtA^C173S‐GFP and ArtA^R214A‐GFP cannot process substrates, while replacement of the third residue, ArtA^R253A‐GFP retains some processing activity. These findings support the view that similarities between certain aspects of the structures and functions of the sortases and archaeosortases are the result of convergent evolution.

    

   more » « less
5. Comprehensive phylogenetic analysis of the ribonucleotide reductase family reveals an ancestral clade

   https://doi.org/10.7554/eLife.79790  

   Burnim, Audrey A ; Spence, Matthew A ; Xu, Da ; Jackson, Colin J ; Ando, Nozomi ( September 2022 , eLife)

   Billions of years ago, the Earth’s atmosphere had very little oxygen. It was only after some bacteria and early plants evolved to harness energy from sunlight that oxygen began to fill the Earth’s environment. Oxygen is highly reactive and can interfere with enzymes and other molecules that are essential to life. Organisms living at this point in history therefore had to adapt to survive in this new oxygen-rich world. An ancient family of enzymes known as ribonucleotide reductases are used by all free-living organisms and many viruses to repair and replicate their DNA. Because of their essential role in managing DNA, these enzymes have been around on Earth for billions of years. Understanding how they evolved could therefore shed light on how nature adapted to increasing oxygen levels and other environmental changes at the molecular level. One approach to study how proteins evolved is to use computational analysis to construct a phylogenetic tree. This reveals how existing members of a family are related to one another based on the chain of molecules (known as amino acids) that make up each protein. Despite having similar structures and all having the same function, ribonucleotide reductases have remarkably diverse sequences of amino acids. This makes it computationally very demanding to build a phylogenetic tree. To overcome this, Burnim, Spence, Xu et al. created a phylogenetic tree using structural information from a part of the enzyme that is relatively similar in many modern-day ribonucleotide reductases. The final result took seven continuous months on a supercomputer to generate, and includes over 6,000 members of the enzyme family. The phylogenetic tree revealed a new distinct group of ribonucleotide reductases that may explain how one adaptation to increasing levels of oxygen emerged in some family members, while another adaptation emerged in others. The approach used in this work also opens up a new way to study how other highly diverse enzymes and other protein families evolved, potentially revealing new insights about our planet’s past. 

   more » « less